Pathway

Mechanisms of Commensal- Specific CD8+ T Cell Differentiation, Restraint and Dysregulation in Intestinal Inflammation

PROJECT SUMMARY Our understanding of immunity largely stems from models of infection with pathogenic microbes. However, the vast majority of microbial-immune encounters occur as a symbiotic relationship with the commensal microbiota. Recently, the contribution of commensal-specific T cells to host physiology has received significant attention. These commensal-specific responses not only control microbiota containment but also promote immune tolerance within the gastrointestinal tract. While commensal-specific CD4+ T cell responses in the lamina propria have dominated models of mucosal immune regulation, these are vastly outnumbered by CD8+ intraepithelial lymphocytes within the epithelium. How CD8+ T cell responses to gut microbiota are primed, differentiate and function under homeostasis has not been addressed. Conversely, aberrant immunity to commensal microbes has been proposed to underlie pathologies of barrier tissues, including inflammatory bowel disease (IBD), where commensal-specific T cells accumulate in blood and intestinal tissues of afflicted patients. A better understanding of the properties and functions of commensal-specific T cell responses is therefore fundamental to studies of tissue immunity in health and disease. Our long term goal is to better understand how commensal-specific T cell responses contribute to barrier tissue homeostasis, and the objective in this application is to investigate the mechanisms regulating induction of commensal-specific CD8+ T cells in homeostasis and how they become dysregulated in IBD. Our rationale for the proposed work is that uncovering these mechanisms has the potential to translate into new therapeutic approaches. Our central hypothesis is that commensal-specific CD8+ T cells develop as functionally restrained intraepithelial lymphocytes (IEL) under homeostasis, but that perturbation of local immune regulation within the intestinal epithelium, in the case of patients with ulcerative colitis, by autoantibody-mediated blockade of integrin avb6 results in aberrant CD8+ effector T cell responses in IBD. Based on strong preliminary data, we will test three specific aims: (1) Determine key antigen-presenting cells (APC) priming SFB-specific CD8⍺β+ IEL. (2) Identify how cell-intrinsic pathways drive differentiation, maintenance and restraint of SFB-specific CD8⍺β+ pIEL. (3) Determine how pathogenic KLRG1+Eomes+ CD8+ T cells arise and contribute to inflammation in murine models of ulcerative colitis Our approach is innovative as it investigates new mechanisms of immunity unique to commensal-specific CD8+ T cell responses. The proposed work is significant because it will establish new insights into the interaction and communication between commensal microbes and immune cells in the gut environment and identify potential targets for therapeutic intervention in conditions of chronic intestinal inflammation.

Staphylococcus aureus metabolic requirements during skin colonization

Project Summary Staphylococcus aureus causes 76% of all skin infections, and yet simultaneously this pathogen asymptomatically colonizes the skin of 8-22% of healthy adults. Since the majority of S. aureus disease is the result of autoinfection from the colonizing strain, and invasive infections often originate from the skin, there is an urgent need to understand colonization mechanisms. In colonizing the skin, S. aureus encounters abundant levels of amino acid derivatives like urocanic acid and 5-oxoproline (OP) that contribute to the skin’s “acid mantle” and have reported anti-Staphylococcal properties. The central hypothesis of this project is that amino acid transport and catabolism is a critical feature of S. aureus skin colonization. To model this environment, we developed a skin-like media (SLM) to assess S. aureus physiology on the human skin surface. We determined the S. aureus transcriptional response using RNAseq and performed metabolomics in SLM, both of which demonstrated that amino acid catabolism genes are upregulated and that amino acids are rapidly consumed. These findings indicate that S. aureus has a skin expression program that enables survival and growth in this harsh environment. In Specific Aim 1, we are investigating S. aureus metabolism of serine, the second most abundant amino acid on human skin. We hypothesize that serine transport and catabolism is critical for S. aureus skin colonization. We will assess growth of mutant strains disrupted in serine pathways in the SLM and during mouse skin colonization. With 13C-tracing experiments we will investigate serine flux in S. aureus using metabolomics. We will determine serine transport mechanisms using bioinformatic guided targets and serine analogues. In Specific Aim 2, we will assess S. aureus resistance to toxic skin metabolites. OP is abundant on human skin and is known to be deleterious to bacteria. Our preliminary metabolomics studies indicate that S. aureus metabolizes OP in SLM, and we have identified a putative oxoprolinase (genes SAUSA300_1566-1561) that is upregulated on skin. We hypothesize that the detoxification of OP contributes to S. aureus survival on the skin. We will construct mutants in the 1566-1561 locus and test their contributions to OP metabolism in SLM with growth and metabolomics experiments. We will also investigate OP transport and test mutant strains in our mouse skin colonization model. In Specific Aim 3, we will identify new determinants of S. aureus skin colonization using TnSeq. We have developed an improved TnSeq library preparation and analysis protocol, and in our preliminary studies we performed TnSeq in SLM and in our mouse skin colonization model. We will evaluate pathway hits, such as respiration and fermentation, and aspartate metabolism targets by testing constructed mutants during SLM growth and in the mouse model. Novel hits will be validated with follow-up genetic experiments and 13C-tracing experiments. Collectively, the proposed studies will advance our knowledge of S. aureus colonization and adaptation to the skin environment.

Weak Cell Adhesion is a Prognostic Signature of Invasive Cancer

Project Summary Despite early detection, low-grade and localized breast cancers such as ductal carcinoma in situ (DCIS) can relapse in up to 20% of cases despite standard of care. For DCIS, relapse affects over 12,000 U.S. women annually and has increased 60% in the last 40 years. Current diagnostic assessments including histopathological markers often miss early disseminating cells, lack specificity, or cannot distinguish cancer from non-cancer cells in the stroma. Hence there is an unmet need for cancer diagnostic technologies that employ radically different characterization methods. For example, significant physical differences exist between metastasizing and benign breast cancer cells, owing to metastasizing cells detaching from the primary tumor, migrating through the surrounding stroma, intravasating and extravasating, and ultimately engrafting in distant tissues. We recently demonstrated that cancer cells with weaker adhesion migrate faster and metastasize more frequently in murine breast cancer models than strongly adherent cells. In a small pilot study of human breast tumors, we also observed that the abundance of weakly adherent (WA) cells scales with disease severity; subpopulations from invasive carcinomas were the least adherent. However, a subset of DCIS cases displayed much less adhesion, suggesting that these patients may have a tumor subpopulation that progresses to metastatic disease despite standard-of-care treatment. Weak adhesion is a defining physical characteristic of tumors, but to establish their role in initiation, metastasis, and patient outcomes, we will leverage model systems and our newly patented adhesion technology to answer these fundamental questions of cancer biology and clinical translation. To understand the impact of adhesion on cancer progression, we will evaluate the tumor-initiating potential of WA versus strongly adherent (SA) tumor cells in a murine breast cancer model before confirming how weak adhesion advantages cells to cause secondary disease using bioengineered in vitro models. In dissecting the stages of metastasis where WA cells exhibit advantages, e.g., recapitulating stromal niche, transendothelial migration, and tissue-specific colonization, we will identify mechanisms that enable WA cells to thrive and evaluate therapeutic targets that disrupt these pathways. Finally, we will analyze the adhesion profiles of resected tumors and stroma from 80 breast cancer patients with DCIS or invasive disease. Adhesion data will be correlated with conventional assessment methods and ultimately with patient outcomes, e.g., disease-free and progression-free intervals. We anticipate that the DCIS subpopulation that aligns with the adhesion signature of invasive carcinomas will have shorter intervals and survival time. This integrated study design bridges mouse models, mechanistic bioengineering assays, and human samples to clarify the metastatic potential and prognostic value of WA breast cancer cells. Our use of mouse models in this grant is required to study the interactions among tumor cells, immune cells, vasculature, and stromal tissues that drive tumor formation in vivo. Bioengineered in vitro systems lack the complexity to ask such questions and using injected tumor cells is not possible in humans.

Defining Microbial and Host Pathways Driving Asymptomatic C. difficile Colonization Associated with Aging and High-Sugar Diets

SUMMARY Clostridioides difficile infection (CDI) is a leading cause of healthcare-associated diarrhea, with rising incidence in community settings and a growing burden of asymptomatic colonization. Asymptomatic car- riers, particularly among the elderly and individuals consuming high-sugar diets, represent a critical but underexplored reservoir for transmission and disease progression. This proposal introduces novel, anti- biotic-independent mouse models demonstrating that both dietary sugar and aging independently pro- mote asymptomatic C. difficile colonization. We hypothesize that these factors disrupt colonization re- sistance (CR) through distinct but overlapping microbial, metabolic, and immune pathways. In Aim 1, we will define how traditional and emerging dietary sugars alter the gut environment to permit C. difficile colonization using in vitro bioreactors and in vivo models. Aim 2 will identify age-associated changes in microbiota and mucosal immunity that impair CR, using longitudinal studies and fecal micro- biota transfer. Aim 3 will functionally validate C. difficile genes upregulated during asymptomatic carriage using CRISPR-Cas9 mutants in both sugar- and age-induced models. This integrative, multi-omics approach will uncover the mechanisms enabling asymptomatic colonization and identify microbial and host targets for intervention. The findings will inform microbiome-based strat- egies to prevent CDI in vulnerable populations and shift current paradigms in CDI risk assessment and prevention.

The Role of the Intestinal Microbiota in Sepsis Mortality

Project Summary/Abstract Sepsis is a life-threatening condition characterized by a dysregulated host response to infection that can cause multi-organ damage and death. As the leading cause of in-hospital mortality, sepsis mortality rates reach up to 50%, and account for approximately 270,000 deaths and $38 billion annually in health care costs in the United States. Notably, patients with similar medical backgrounds can have vastly different sepsis outcomes— some survive with medical treatment while others die. The reasons for this dichotomy are unknown but is seen across all forms of bacterial bloodstream infections, is not specific to any strain-level differences in the infecting pathogen and cannot be explained by human genetic differences. Human microbiota studies suggest that gut microbial dysbiosis is associated with sepsis mortality and that these alterations influence gut barrier breakdown, leading to gram-negative bacteremia—one of the most common causes of sepsis and mortality. However, there are a lack of studies that investigate the causal role of the intestinal microbiota in sepsis mortality. This K08 proposal will elucidate the role of the intestinal microbiota in sepsis mortality. Utilizing the well- established murine model of sepsis by intraperitoneal injection of lipopolysaccharide (LPS), we combine microbiota taxonomic sequencing and metagenomics, advanced bioinformatic techniques and prediction modeling, with knowledge of mucosal immunity and germ-free mouse systems to characterize the microbiota features and members that correlate with, predict, and cause sepsis mortality. This proposal is organized into two specific aims: (1) identify baseline stool microbial features associated with and predictive of sepsis outcomes and (2) determine how colonization with immunostimulatory microbes heightens sepsis mortality. In this work, I will holistically characterize the host immunologic and microbiota features that are associated with and predictive of mortality and experimentally identify microbes and microbial pathways that cause death in our model. These findings will reveal new microbial and host biomarkers of sepsis mortality and identify novel targets for sepsis prevention and treatment to reduce the overall mortality rate of this deadly disease. My long-term goal is to become an independent physician-scientist who integrates cutting-edge computational methods with experimental biology to identify predictive biomarkers of disease onset and outcomes, investigate how they influence disease processes, and develop novel therapeutic and preventive strategies to improve patient care. This proposal details specific research aims and a structured career development and training plan that will allow me to acquire focused, in-depth and multidisciplinary training under the guidance of an internationally recognized team of experts in clinical infectious diseases, host-microbiota interactions, immunology, immunometabolism, and computational biology. The knowledge generated will address the fundamental role of the microbiota in sepsis outcomes and inform future preventative and therapeutic strategies that will lower the sepsis mortality rate worldwide.

Eosinophils promote persistence and transmission during Bordetella spp. infections

ABSTRACT Despite widespread vaccination, Bordetella spp., the causative agents of whooping cough, continue to circulate globally. Resurgent outbreaks contribute to significant healthcare burdens and costs estimated up to $79 million annually. This persistence and reemergence highlight a critical need for new therapies and prevention methods. Our laboratory investigates bacterial and host drivers that enable Bordetella success, defined as enhanced persistence, reinfection, and transmission. We have identified the Bordetella sigma factor BtrS as a regulator of immunosuppressive pathways that modulate eosinophil function. Leveraging genetically tractable Bordetella strains, advanced murine models, and immunological tools, we are uniquely positioned to dissect how eosinophils contribute to respiratory bacterial infections. Our preliminary data reveal that eosinophils promote Bordetella persistence. Our results also show that the anti-inflammatory cytokine IL1 receptor antagonist (IL1Ra) also contribute to persistence. However, the contribution of eosinophil-derived immunosuppressors remains unclear and will be investigated in Specific Aim 1. Moreover, we have evidence that eosinophils are required for nasal shedding, through mucus enhancement, and paroxysmal coughing, via exacerbation of bronchoconstriction, during Bordetella spp. infection, two key metrics of transmission. The eosinophil-effectors that promote shedding, coughing, and transmission, will be investigated in Specific Aim 2. Based on our data, we hypothesize that eosinophils contribute to Bordetella pathogenesis by (1) promoting persistent infection and (2) enhancing transmission through mucus-driven shedding and cough reflex induction. This proposal will test this hypothesis through two specific aims: Aim 1: Delineate the immunosuppressive role of eosinophils in modulating host responses and enabling Bordetella persistence. Aim 2: Define the mechanisms by which eosinophils facilitate Bordetella spp. transmission. By reframing eosinophils as active modulators of bacterial pathogenesis, this research challenges traditional views of eosinophils as terminal effector cells and positions them as novel targets for therapeutic intervention, that might be applicable to other mucosal pathogens. The outcomes will contribute to our understanding of eosinophil biology in infection and may lead to innovative strategies to halt bacterial persistence and transmission.

Targeting disulfidptosis in cancer: mechanisms and preclinical translation

Project Summary Studying regulated cell death is critical for our understanding of cellular homeostasis and tumor suppression. We recently discovered disulfidptosis as a new form of regulated cell death induced by disulfide stress under NADPH-depleting conditions in SLC7A11-high cancer cells. However, in contrast to our deep understanding of other cell death modalities such as apoptosis and ferroptosis, the molecular and metabolic underpinnings of disulfidptosis, along with its therapeutic implications, remain largely unexplored. The objectives of this application are to elucidate the mechanisms underlying disulfidptosis and to therapeutically target this form of cell death in SLC7A11-high cancers. The proposed studies will make extensive use of human cancer cell lines and integrated human cellbased molecular analyses, including metabolomics, proteomics, CRISPR screening, and biochemical studies, to define the metabolic and signaling mechanisms governing disulfidptosis. In addition, select in vivo studies are incorporated in the therapeutic validation components of the project, where tumor growth response, systemic drug exposure and tolerability, tumor microenvironmental influences, and host immune/stromal interactions must be evaluated in an organismal context to ensure translational rigor. Alternative in vitro systems such as organoids may provide useful complementary information on tumor-intrinsic responses, but they cannot fully recapitulate the systemic metabolic stress, pharmacologic exposure, and organism-level therapeutic efficacy required for these studies. It is expected that our proposed studies will reveal novel mechanisms underlying disulfidptosis and identify effective therapies to induce this form of cell death in SLC7A11-high cancers. Our proposal is highly innovative because it focuses on a previously unexplored cell death pathway in cancer therapy. Our proposed studies will have significant impact on both our understanding of the fundamental mechanisms of disulfidptosis and our ability to target this cell death pathway in cancer treatment.

Integrins α4β7 in Leukocyte Rolling in Shear Flow, Firm Adhesion, and Therapy

Abstract. Integrin α4β7 facilitates leukocyte migration to sites of infection and autoimmune disease, making it an important therapeutic target for ulcerative colitis and Crohns disease. However, the currently approved antibody drug vedolizumab targeting α4β7 has limited efficacy. This proposal seeks mechanistic understanding of how α4β7 mediates rolling and firm adhesion of leukocytes during extravasation as well as how therapeutically relevant antibodies modulate α4β7 function to improve drug design. Unlike most integrins, α4β7 mediates rolling adhesion on its ligand MAdCAM. α4β7 can also mediate firm adhesion like α5β1. Integrins typically equilibrate between two low-affinity closed conformations and a high-affinity open conformation. Ligand binding is intimately coordinated with conformational change. During rolling adhesion, receptor-ligand bonds must rapidly form beneath rolling cells as cells are torqued by shear flow onto the substrate. Bonds must also rapidly dissociate at the upstream tethers to the substrate due to hydrodynamic force applied to the cell. To enable their function in rolling adhesion, we hypothesize that α4β7 ligand binding and dissociation and conformational change kinetics are faster than those of other integrins like α5β1 and that α4β7's pathways for conformational change may also differ. We propose that activation of the actin cytoskeleton in the transition from rolling to firm adhesion stabilizes α4β7 in a high-affinity state. Aim 1 will determine high-resolution structures of unliganded α4β7 and its complexes with MAdCAM or medically relevant antibodies using cryo- EM. These structures will reveal how these integrins recognize their ligands, the conformational changes due to ligand binding, and potential structural specializations that enable α4β7 to mediate rolling adhesion. The binding epitopes and conformational specificities of activating antibodies to the β7 subunit will also be defined. The structure of α4β7 bound to vedolizumab will resolve the contention around how it blocks MAdCAM binding. Aim 2 will quantitatively define the mechanisms by which α4β7 mediates both rolling and firm adhesion to improve therapies for inflammatory bowel diseases. Ligand affinity and binding kinetics of α4β7 stabilized in different conformations will be measured as well as single-molecule conformational change rates when bound and unbound to ligand. The effect of mutations that stabilize rolling or firm adhesion will be used to identify parameters important for each adhesion type. The tensile force and bond lifetimes during rolling and firm adhesion will be quantified at the single-molecule level. Together, our studies will enhance our structural, biochemical, and mechanical understanding of α4β7-mediated rolling and firm adhesion and will provide structural and functional information that can be utilized in the development of more effective therapies for inflammatory bowel diseases and multiple myeloma.

Dissecting the role for astrocytes in mediating adverse outcomes of maternal immune activation.

Prenatal infections cause maternal immune activation (MIA), a major risk factor for several neurodevelopmental disorders, including schizophrenia, autism spectrum disorders (ASD), and attention deficit hyperactivity disorder (ADHD). Consequently, elucidating the mechanisms by which MIA alters brain function is critical for understanding the pathophysiology of these disorders and developing effective treatments. While the effects of MIA on neurons and microglia have been extensively studied, the impact of MIA on astrocytes, key regulators of brain physiology and homeostasis, remain unknown that significantly impedes our understanding the mechanisms of MIA-induced neurobehavioral abnormalities. To address this major knowledge gap, we conducted pilot studies that suggest that MIA increases impulsivity-like behaviors and amphetamine-induced hyperactivity and enhances extracellular levels of glutamate (GLU) and dopamine (DA) in the dorsal striatum (DS). MIA also increased pro-inflammatory signatures of astrocytes, including up- regulation of the Nuclear Factor kappa B (NF-κB) pathway and increased GFAP immunoreactivity in DS astrocytes. Collectively, these novel findings support our overarching hypothesis that MIA increases astrocyte reactivity, leading to increased gliotransmission (e.g., GLU), which in turn enhances DS DA release and DA- dependent behaviors. To test this hypothesis, we will leverage the expertise of the research team in molecular, physiological and neurobehavioral approaches and conduct the following Specific Aims: In Aim 1, we will identify the MIA-induced cellular and physiological changes characteristic of astrocyte reactivity. In Aim 2, we will determine the circuit mechanisms by which MIA increases DA signaling. In Aim 3, we will identify the molecular mechanisms whereby reactive astrocytes contribute to MIA-induced cellular and behavioral abnormalities. These studies will enhance the current understanding of the effects of MIA on brain functions and generate new insight into potential treatment strategies for MIA-associated neurodevelopmental disorders.

Linking Single-Cell Transcriptomic, Morphological, and Temporal Signatures of Vulnerability in Neurodegeneration

Neurodegeneration involves complex cellular phenotypes and molecular changes that vary widely among the cells of the nervous system. Current methodologies permit either detailed molecular profiling (e.g., single-cell transcriptomics) or functional phenotyping (e.g., live imaging of neuronal activity), but not both in the same cells. Thus, it is difficult to directly link a neuron's functional state or fate with its gene expression profile. To address this limitation, we developed an innovative technology, VISTA-FISH (Video Imaging with Spatial- Temporal Analysis by FISH), that couples prospective live-cell imaging with high-resolution spatial transcriptomic profiling of the same cells. This approach enables in situ comparisons of gene expression in neurons that exhibit divergent behaviors or outcomes. Using VISTA-FISH, we will profile iPS-derived human neurons to link single-cell gene expression, morphology, and temporal phenotypes to study molecular pathways driving resilience as well as susceptibility. After exposing neurons carrying TDP43 and C9orf72 mutations to a stimulus inducing TDP43 aggregation, we will jointly record TDP43 localization and neuron activity using live-cell microscopy, then measure single-cell gene expression of the same cells (Aim 1). We will also combine live-cell measurements of TDP43 half-life with CRISPR screening and single-cell gene expression (Aim 2). These rich datasets will enable us to determine transcriptomic changes associated with differences in protein aggregation, protein synthesis, and protein degradation in individual cells, providing an unprecedented molecular perspective on factors responsible for vulnerability and resilience to neurodegeneration.

Metabolic Assessment of Metformin in Pregnancy (MoM-P)

PROJECT SUMMARY The objective of the “Metabolic Assessment of Metformin in Pregnancy “(MoM-P) proposal is to assess the physiological effect of metformin on maternal and neonatal metabolism during pregnancy in individuals developing gestational diabetes (GDM). Metformin is increasingly being used for medical treatment of GDM not adequately treated with nutrition and physical activity. There is inconsistency among various organizations (Society for Maternal Fetal Medicine, American College of Obstetrics and Gynecology and the American Diabetes Association) as to metformin’s role in the medical management of GDM. We will examine the metabolic action of metformin in GDM pregnancies and effect on mothers and their offspring. We plan to recruit 50 participants from Massachusetts General Hospital (MGH) for Specific Aims 1, 2 and 3 and 100 participants from Ohio State University college of Medicine (OSUCOM) for Specific Aims 2 and 3. Participants for the study will have been diagnosed with GDM requiring medical management of GDM as part of the DECIDE multicenter randomized controlled trial. The primary site for DECIDE is OSUCOM, with Dr. Mark Landon as the PI. The MoM-P study will recruit participants from the DECIDE trial at MGH and OSUCOM. The MoM-P study aims are: Aim 1: To establish metformin’s effects on endogenous (primarily hepatic) glucose production (EGP) and insulin sensitivity in late pregnancy. We hypothesize that metformin does not lower EGP in pregnancy and hence the need of additional insulin in the medical management of GDM. We will perform infusion of a stable isotope of glucose (6,6 2H2 glucose) to estimate EGP and a HOMA-IR prior to initiation of medical management and again at 37 weeks gestation. Aim 2: Metformin increases GDF15 levels in human GDM pregnancy and is associated with lower nutrient intake, gestational weight gain (GWG) and increased resting energy expenditure (REE). We hypothesize that metformin increases GDF15 concentrations which lead to GI upset, lower caloric intake/GWG and increases REE. In DECIDE participants randomized to metformin vs. insulin, we will measure GDF15 and examine the relationship to ASA-nutrition records, REE with indirect calorimetry and maternal body composition using air displacement plethysmography (ADP) prior to initiation of medication and again at 37 weeks. Aim 3: To compare fetal growth and body composition in neonates exposed and unexposed to metformin in utero. We hypothesize that metformin treatment of GDM decreases fetal weight: 1) directly based on metformin’s effect on neonatal metabolism (fetal AMPK and mTOR pathways) and 2) indirectly by lowering maternal nutritional intake, fat free mass (FFM) and increasing maternal REE, resulting in decreased neonatal FFM and increased fat mass in childhood. In DECIDE participants, we will measure neonatal body composition with 72 hours of delivery using pediatric ADP and a planned follow-up of children at 2 years in the DECIDE protocol with estimates of male and female children’s body composition.

COCHLEAR SIGNALING MEDIATED BY HENSEN’S CELLS

PROJECT SUMMARY/ABSTRACT The organ of Corti has two types of auditory sensory cells (inner and outer hair cells) surrounded by nearly a dozen different types of supporting cells organized in a very meticulous pattern. Hair cells mediate the mechano-electrical transduction process of the organ of Corti and thus most cochlear auditory research has focused on these sensory cells. In contrast, much less is known about the different types of cochlear supporting cells, even though they likely impact hair cell function. Hensen’s cells are located laterally to the outer hair cell rows and appear to be the only cell type in the cochlear epithelium that expresses TRPA1 channels. These channels are widely known for their role as sensors of tissue damage and inflammation in nociceptive neurons. Not surprisingly, we recently found that Hensen’s cells are main sensors of tissue damage in the cochlear epithelium via the activation of TRPA1 channels (Velez-Ortega et al., Nat Commn, 2023). Additionally, our preliminary data also supports the role of Hensen’s cells in signaling pathways important for the proper innervation of the organ of Corti (aim 1), for the transmission of cochlear damage signals to the brain (aim 2), and for the regulation of hearing sensitivity after acoustic trauma (aim 3). Thus, here we will explore the hypothesis that TRPA1- mediated signaling pathways in the Hensen’s cells are required for the proper innervation and auditory function of the organ of Corti. In Aim 1 we will perform a detailed comparison of the morphology and synapses of afferent cochlear neurons of wild-type and Trpa1-/- mice at several developmental stages (using immunolabeling, confocal microscopy, STED microscopy, and electron microscopy) to assess the role of TRPA1 activity on the postnatal refinement of the cochlear innervation. Aim 2 will evaluate whether the afferent type II spiral ganglion neurons (SGN) can be activated downstream of TRPA1 channel gating in Hensen’s cells by testing responses of neonate and adult type II SGN to TRPA1 agonists (via live-cell time-lapse calcium imaging and patch clamp recordings of type II SGN dendrites). Aim 3 will test the impact of TRPA1 signaling in Hensen’s cells to the operating point of the cochlear transducer (via the recording of cochlear microphonics) and to cochlear tuning (via the recording of ABR tuning curves). This study is significant because it will contribute to our understanding of the cellular (Hensen’s cells plus type II SGN) and molecular (TRPA1 channels) mechanisms of the elusive cochlear nociceptive pathway. In addition, given that the loss of TRPA1 channels does not affect hearing thresholds in mice, we believe that undiagnosed deficits in TRPA1-dependent responses in the human population could represent a hidden susceptibility for cochlear damage after noise exposure or other insults.

Hepatotoxicity of Legacy and Replacement PFAS: Role of BRUCE-Mitochondrial Interactions

Epidemiological studies have shown a strong association between exposure to PFAS (Per- and Poly- fluoroalkyl Substances) and liver toxicity. Particularly, legacy C8-PFAS members, PFOS (perfluorooctane sulfonate) and PFOA (perfluorooctanoic acid), are highly toxic, with PFOS estimated to be approximately 10 times more toxic than PFOA in ecotoxicity models. Consequently, PFAS replacements such as GenX and PFBS are marketed as safe alternatives, although growing evidence indicates that these substitutes also exhibit toxic effects. Lab animal model studies have shown hepatotoxic effects of both legacy and replacement PFAS members, characterized by Metabolic dysfunction-associated steatotic liver disease (MASLD) and its severe form Metabolic dysfunction- associated steatohepatitis (MASH), the two chronic liver diseases affecting an estimated 80-100 million Americans. The broader objective of this project is to understand the underlying mechanisms of PFAS hepatotoxicity in MASLD/MASH. In this context, our initial studies have shown that PFAS exposure of mice downregulates hepatic BRUCE, an autophagy inhibitor, resulting in development of MASLD in WT, and more severe MASLD and even progression to MASH in BRUCE liver-knockdown (BKO) mice. Using primary hepatocytes, we found PFAS-induced BRUCE reduction compromised mitochondrial (mt) functions (respiration, fatty acid oxidation/FAO, and ATP production) and suppressed mitophagy in WT and more so in BKO mice. Pharmacological restoration of mt function in mice prevented PFAS-induced MASLD/MASH. Guided by these compelling preliminary data and scientific premise, we hypothesize that PFAS degradation of BRUCE in hepatocytes induces excessive autophagy (resulting in cytotoxicity) and inhibits mitophagy (resulting in accumulation of damaged mitochondria), leading to release of mtDAMPs to activate inflammation/ fibrosis, thereby facilitating progression from MASLD to MASH. We will test this by three specific aims. Aim 1 (ex vivo) is to determine the human-relevant PFAS doses that modulate BRUCE levels for homeostatic vs cytotoxic autophagy and how BRUCE in turn regulates autophagy. Aim 2 (ex vivo) will investigate BRUCE-driven mitophagy pathway specific to PFAS exposure at human-relevant doses. Aim 3 (ex vivo and in vivo) will involve ex vivo simulation experiments to characterize the role of PFAS-induced, BRUCE-dependent hepatocyte- released mt DAMPs in activation of immune and fibrogenic cells using co-culture assays. Next, we will perform in vivo intervention to validate the role of PFAS-damaged mitochondria in driving MASH progression in mouse models. Furthermore, human relevance of the delineated mechanisms will be ascertained and validated using iPSC-derived human liver organoid system. Impact: This project will advance our understanding of autophagy/mitophagy-centric mechanisms with therapeutic potential in the context of PFAS-induced liver disease MASLD/MASH.

Specific Affinity Requirements for Antibody Somatic Hypermutation

PROJECT SUMMARY Antibodies diversify through two distinct pathways. The first involves the combinatorial assembly of immunoglobulin (Ig) heavy and light chain variable region (V) exons, forming the antigen recognition domains of the B cell receptor (BCR), which is initially expressed as IgM on immature B cells. The second diversification pathway is somatic hypermutation (SHM) of V exons in germinal centers (GCs). In this setting, B cells that acquire mutations enhancing affinity for antigen receive limited cognate T cell help and are selected for clonal expansion, leading to affinity maturation. These primary and secondary diversification systems work together to generate protective antibody responses. The primary, or pre-immune, repertoire provides the foundation for initial antigen recognition. SHM and affinity maturation refine these baseline specificities. While it is well established that SHM improves affinities already present in the primary repertoire, this project explores the hypothesis that SHM can also generate new specificities in B cells that initially lack measurable antigen recognition. This process, termed affinity birth, may enable access to otherwise excluded V gene segments and expand the landscape of antibody evolution. This hypothesis will be tested through two specific aims: (i) To elucidate the extent of SHM-mediated Ig diversification in non-specific or bystander B cells. And, (ii) to define parameters that influence SHM-mediated antibody affinity birth. The significance of this work lies in its potential to reveal previously unappreciated flexibility in the antibody diversification process and to uncover modifiable factors that influence the emergence of new specificities. The proposed studies are innovative in suggesting that B cells possess intrinsic capacity to undergo SHM and selection regardless of their initial antigen specificity. This research may advance understanding of how germinal centers support antibody evolution and inform strategies to design vaccines that anticipate emerging pathogens.

Mechanisms and consequences of cerebrovascular dysfunction in preeclampsia

PROJECT SUMMARY/ABSTRACT Preeclampsia (PE) is a common hypertensive disorder of pregnancy that causes significant maternal and fetal morbidity and mortality worldwide. PE women are at a high risk of stroke, including intracerebral hemorrhage, during the peripartum period, suggesting the sequelae of PE adversely impacts the cerebral circulation to promote hemorrhage. In addition, women with severe early-onset PE are at an 85-fold increased risk of death from intracerebral hemorrhage, importantly suggesting severity of disease promotes greater vulnerability of the cerebral circulation to degradation and rupture. However, the consequences of PE extend far beyond pregnancy and are associated with excessive cardiovascular and cerebrovascular disease risk later in life. Women with previous pregnancy complicated by PE can develop cognitive impairment as early as in their 30’s and 40’s, suggesting PE predisposes the brain to early-onset cognitive impairment. Studies have shown that formerly PE women have changes in gray matter volume and increased white matter lesion burden that occurs as a function of time from pregnancy, suggesting that PE continues to progressively damage the brain long after the affected pregnancy. Thus, our overall goal is to elucidate mechanisms by which women with PE are at risk of intracerebral hemorrhage in pregnancy and cognitive decline later in life. Our preliminary studies found greater vascular degradation, hematoma and cerebral edema in a model of severe PE that was associated with vascular inflammation and microglia activation (neuroinflammation). In addition, we found endothelial dysfunction and diminished neurovascular coupling in PE rats that persisted 5 months postpartum. Impaired neurovascular coupling is well-recognized as an underlying contributor to cognitive decline. These effects in postpartum animals with previous exposure to PE were associated with memory impairment that was not present in the pregnant state, suggesting neurovascular dysfunction precedes cognitive decline. Our central hypothesis is that the sequela of PE accelerates hypertension-induced cerebrovascular dysfunction that predisposes to intracerebral hemorrhage during pregnancy and its persistence postpartum results in early-onset cognitive decline. We will therefore elucidate mechanisms by which PE accelerates vascular degradation and worsens outcome from hemorrhagic stroke, probing pathways involved in oxidative degradative processes using multi-omics and multivariate analysis (Aim 1). We will also determine underlying molecular mechanisms that cause persistent cerebral microvascular dysfunction and cognitive decline postpartum, including oxidative stress-induced BBB leakage and persistent neuroinflammation that drives potassium channel dysfunction, reduced neurovascular coupling and neurovascular uncoupling (Aim 2). We will also use machine learning approaches together with multi-omics and outcome measures to identify factors and cellular pathways that are most impactful for prediction of intracerebral hemorrhage and cognitive impairment. The ability to predict and prevent devasting neurovascular disorders associated with PE has the potential to have long-lasting impacts on the lives of women with PE.

Role of stress signals in the pathogenesis of pulmonary veno-occlusive disease

PROJECT SUMMARY/ABSTRACT Pulmonary veno-occlusive disease (PVOD) is a subclass of pulmonary hypertension characterized by preferential remodeling of the pulmonary venules and capillaries, and currently, there are no efficacious drug therapies. The clinical presentations and the radiographic findings of PVOD are indistinguishable from PAH, and therefore, it is often misclassified as PAH. However, the application of PAH therapeutics to PVOD patients leads to life-threatening pulmonary edema, thus, there is a critical need for diagnostic methods that accurately differentiate PVOD from PAH. Genetically, PVOD is associated with biallelic loss of function (LOF) mutations in the EIF2AK4 gene encoding GCN2. GCN2 phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α), shuts down protein synthesis, and activates the integrated stress response (ISR). However, the molecular mechanisms connecting the loss of GCN2 with pulmonary vascular remodeling are poorly understood. Recent studies find that biallelic EIF2AK4 mutations are identified in ~9% of PAH patients. Conversely, heterozygous mutations in the BMPR2 gene, a leading cause of PAH, have been reported in PVOD patients. These results suggest that (i) PVOD and PAH share some of the pathophysiological mechanisms, and (ii) the presence of EIF2AK4 or BMPR2 mutations does not provide an accurate genetic diagnosis for PVOD. The long-term goal of this proposal is to elucidate the pathophysiological mechanisms involved in remodeling not only pulmonary arterioles but also venules and capillaries and develop those pathways as potential therapies for POVD. It has been observed that cancer patients administered with the chemotherapeutic agent mitomycin-C (MMC) rapidly develop PVOD. Rats administered with MMC develop PVOD-like phenotypes, including right ventricular (RV) hypertrophy, increased RV systolic pressure, and pulmonary vascular lesions in arteries and veins. We found that Rad51, an essential enzyme for double-strand DNA break repair, associates with VE-Cad in the vascular endothelium; however, upon MMC treatment, Rad51 and VE-Cad complex (VRC) were released into the circulation, resulting in increased vascular permeability and reduced barrier integrity. MMC treatment also mediates the depletion of GCN2, which recapitulates the genetic cause of PVOD (LOF EIF2AK4 mutations). Based on these data, this proposal will test the hypothesis that the vascular remodeling in PVOD involves (i) the release of VRC, (ii) the aberrant protein synthesis due to the activation of ISR, and (iii) the mechanism of maladaptive ISR activation. Finally, we will explore the potential application of the circulating VRC as a blood biomarker for PVOD.

The Pyruvate-Lactate Metabolic Axis in Heart Failure and Recovery

PROJECT SUMMARY/ABSTRACT Heart failure (HF) is a leading cause of mortality worldwide. The metabolism of the failing heart is commonly characterized by increased glucose uptake, glycolytic dependence, and reduced oxidative phosphorylation. We previously demonstrated that blocking glucose oxidation is sufficient to cause hypertrophy and subsequent HF. Additionally, our preliminary data shows that an altered pyruvate-lactate metabolic axis may be pivotal in human HF. Research investigating both the mechanistic regulation and biological roles of the pyruvate-lactate metabolic axis in cardiac metabolism during HF and cardiac recovery is warranted and also has the potential to identify novel druggable pathways to target for future pharmacological approaches. The overall objective of this application is to test the hypothesis that impaired pyruvate oxidation is a cardinal feature of HF in humans and animal models and that myocardial recovery is tightly coupled to normalization of the pyruvate-lactate metabolic axis. We will quantify the pyruvate-lactate metabolic axis in human HF and myocardial recovery (Aim 1). Next, we will determine the essentiality of the pyruvate-lactate metabolic axis for HF and cardiac recovery (Aim 2). Lastly, we will define cell-autonomous mechanisms that regulate the pyruvate-lactate axis in HF and recovery (Aim 3). These experiments will allow us to identify patterns of metabolic alteration in the pyruvate-lactate axis and molecular pathways during HF and myocardial recovery. Understanding the role of pyruvate and lactate metabolism in HF and myocardial recovery is cutting-edge research. Our unique access to human HF myocardium from patients administered stable isotope-labeled glucose or lactate to quantitate pyruvate metabolism in HF and recovery is state-of-the-art and will likely help us reveal new fundamental mechanisms of cardiac metabolism and expedite the successful translation of therapeutics being validated in various models of HF and recovery.

Bridging Local and System-Wide Autoreactive, Extrafollicular B Cell Signatures in a TLR7-Driven Model

Project Summary A substantial body of literature has described the development of autoreactive humoral responses in the context of autoimmune disease and recently discerned an exciting new avenue for investigation. While early work focused on canonical mechanisms of activation through the germinal center (GC) response, recent studies have found GC infrastructure to be dispensable for the onset of chronic autoimmunity. It has become clear that an alternative pathway of B cell activation, the extrafollicular (EF) pathway, can drive the onset of new autoreactivity in multiple human disorders including rheumatoid arthritis and systemic lupus erythematosus (SLE). In comparison to the GC pathway, the EF pathway represents a less stringent method for B cell activation, leads to accelerated antibody-secreting cell (ASC) formation, and thus has a higher propensity for the production of autoreactive B cell effectors and ASCs. Recently, our group has identified a similar skew toward the EF response in the context of severe viral infection, tied to acute tolerance loss, increased disease severity, and complicated recovery from infection. These findings highlight how further study of the EF response is crucial to our understanding of autoimmune induction across multiple areas of disease. Toll-like receptor 7 (TLR7) stimulation has been identified as a key contributor to EF B cell development in SLE, and several studies have now linked TLR7 overstimulation to chronic autoimmune disease. While EF effector B cell populations have now been identified in both murine models and humans, substantial gaps in our knowledge remain to be answered concerning i) the origins of these cells and ii) the system-wide and microenvironmental signaling and organization that drive this differentiation pathway. We propose to address these gaps, here, by utilizing a TLR7 agonist (R848) in a murine model to characterize the autoreactive response within the blood and draining lymph node through innovative high-throughput analytical techniques. Systemic shifts in proteomic signatures and immune cell phenotype will be monitored in the blood throughout the induction of autoreactivity, using novel applications of machine-learning based classification. These signatures will then be connected to developing inflammatory microenvironments identified within the draining lymph node by applying a customized set of software tools to spatial transcriptomic data. This work will deepen our understanding of the immunologic mechanisms by which the EF pathway can lead to “run-away” autoreactive B cell development, with the added potential for identification of early blood-based biomarkers for this developing autoreactivity. The above proposed work will provide an ideal training opportunity for the candidate to develop experience with advanced immunologic laboratory techniques, rigorous bioinformatic analysis, a systems-level view of immunology, and scientific communication. The Woodruff and Sanz Labs are highly experienced within the autoimmune disease space with extensive experience with the required techniques and established routes for clinical collaboration to act on these findings.

Effects of Apolipoprotein A4 on Lipid Metabolism via Sympathetic Regulation

Obesity increases the risks and progression of hypertriglyceridemia, metabolic dysfunction- associated steatotic liver disease (MASLD), and cardiovascular diseases. Previous studies demonstrate that a single injection of apolipoprotein A4 (APOA4) elevates sympathetic neural activity and fatty acid β-oxidation in adipose tissues; and consistent infusion of APOA4 in obese mice fed a high-fat diet lowers fat mass, reduces hypertriglyceridemia, elevates brown adipose tissue thermogenesis, and attenuates steatosis and enhances sympathetic neural activity in the liver. This project hypothesizes that APOA4 reduces hypertriglyceridemia by regulating lipid metabolism through sympathetic stimulation in adipose tissues (Specific Aim 1) and sympathetic action in the liver (Specific Aim 2). The role of sympathetic action via the neurotransmitter norepinephrine and adrenergic receptor-mediated pathways will be investigated, and their necessity in APOA4-mediated lipid metabolism will be tested. A strength of this project is the interdisciplinary collaboration between investigators with established successful collaboration and publications. The project will provide physiological, molecular, and neurochemical mechanisms underlying how APOA4 differentially regulates metabolism through sympathetic activation in various types of adipose tissues and the liver in male and female obese mice. Findings would provide impetus to develop unique, novel, targeted therapeutic applications against hypertriglyceridemia and MASLD. Importantly, this project will expose undergraduates and graduate students to meritorious research, provide students with hands-on biomedical research experience, and strengthen research environment at R15 eligible institutions.

Multiplex single-cell chemical genomics to identify small molecule modulators of tumor cell-intrinsic immunogenicity in glioblastoma

PROJECT SUMMARY/ABSTRACT Glioblastoma multiforme is the most common and aggressive primary brain cancer. Despite a multimodal treatment regimen of surgical resection, chemotherapy, radiotherapy, and tumor-treating fields, most patients succumb to the disease within two years of diagnosis. Cancer immunotherapy strategies have emerged as a powerful tool for treating aggressive solid tumors such as melanoma and non-small cell lung cancer. However, current strategies have led to low response rates in glioblastoma, resulting from its low immunogenicity. The proposed research program aims to identify small molecules capable of increasing the immunogenicity of glioblastoma cells, focusing on altering gene expression programs associated with recognition by the immune system and the ability of cytotoxic immune cells to target glioblastoma for destruction. We will use highly multiplex chemical transcriptomic profiling to determine the molecular consequence of exposing glioblastoma neurosphere models to 3,792 small molecules, targeting the majority of cellular activities and clinically relevant drug targets as well as a collection of previously identified immunomodulators. We will then determine how each exposure alters the expression of gene programs associated with tumor cell immunogenicity and response to therapy, including the expression of genes associated with the recognition by the immune system and those associated with immune checkpoints, as well as programs more broadly correlated with resistance to anti-cancer therapies. Chemical hits that meet specific criteria will be subjected to a medicinal chemistry review to further classify compounds by their suitability for treating malignancies in the brain. We will then screen chemical hits to determine their ability to modulate immune-mediated tumor cell killing using tumor- immune cell co-culture. Lastly, we will leverage gene editing and flow cytometry to validate hits based on on- target molecular effects and further refine the mechanism of action by inspecting the ability of drugs to modulate immunogenic programs at the protein level. Our chemical genomics screens aim to provide crucial information regarding the link between pathway activity and immunomodulation in GBM, a critical step to guide future efforts in GBM immunotherapy. More broadly, our study will establish single-cell chemical genomics as a scalable platform for phenotype-based screening for preclinical prioritization of chemical modulators of complex transcriptional phenotypes and provide a framework for hit prioritization, establishment of pipeline robustness and hit validation in the context of single- cell chemical genomics screens.

Breaking Tolerance: Trichloroethylene Provides Survival Signals to Autoreactive CD4s in the Liver

PROJECT SUMMARY The industrial solvent and widespread environmental contaminant, trichloroethylene (TCE) has been linked to autoimmune disease in humans. How TCE impairs tolerance (i.e., unresponsiveness) to self-antigens leading to autoimmunity has not been explored. Autoimmune diseases (ADs) are a class of disorders that affect many different organs and tissues. However, all autoimmune diseases share a feature in common which is the ability of potentially pathogenic autoreactive cells to evade deletion. During early life, peripheral CD4+ cells are primarily comprised of recent thymic emigrants (RTE) which home to the liver. The liver is known to efficiently retain and tolerize self-reactive CD4s to where they are functionally unresponsive to their antigen. Thus, the liver is the first checkpoint in the periphery to filter, retain, and enforce tolerance to autoreactive CD4+ RTEs. The liver is also the site of TCE metabolism. Our Aims are designed to test the hypothesis that TCE, through its metabolite TCAH, delivers costimulatory signals to liver CD4 RTEs via CD28, thereby overriding inhibitory CTLA-4 signaling. This disruption promotes the survival of self-reactive CD4 RTEs by impairing CTLA-4-dependent tolerance mechanisms contributing to the development of ADs. This research will significantly advance the fields of toxicology and autoimmunity, where the origins of environmentally induced AD remain poorly understood. Aim 1 will assess TCE’s effects on RTE migration patterns in real-time in transgenic mice. Aim 2 will investigate TCAH-mediated costimulatory signaling in CD4 RTEs in vitro. Successful completion of these studies will determine how TCE alters key tolerance pathways in the liver resulting in a greater proportion of self-reactive effector memory (EM) peripheral CD4s capable of promoting AD.

Pathogenic mechanisms of expanded ZFHX3 in SCA4 cerebellar organoids

Spinocerebellar ataxia type 4 (SCA4) is a disabling neurodegenerative disease characterized by progressive cerebellar ataxia, and the causative GGC-repeat expansion in ZFHX3 (ZHFX3-exp) was just discovered this year by our lab and others. Our research aims to understand how ZFHX3-exp causes SCA4 and to identify molecular therapeutic targets that can be quickly advanced into clinical trials. SCA4 is one of the four poly-glycine diseases that share the presence of neuronal intranuclear inclusion (NIIs) as a disease hallmark. In SCA4, NIIs are positive for ZFHX3, p62 and ubiquitin, indicating the loss of proteostasis as a mechanism of neurodegeneration. In addition, ZFHX3 RNA-gain-of-function may also contribute to neurodegeneration. Beyond this, knowledge of the disease mechanisms that underly SCA4 is extremely limited and there are currently no disease-modifying treatments for SCA4 or other polyG/NII diseases. There are no SCA4 mouse models and because of the high GC content in the repeat expansion complicates the production of SCA4 mouse models. We propose a novel approach to characterizing SCA4 Purkinje cell (PC) pathogenesis using human cerebellar organoids. Our approach allows for rapidly advancing the understanding of the pathogenesis and potential treatments of SCA4. Using cerebellar organoids will enable investigation on functional PCs, cerebellar neurodegeneration and the testing of potential therapeutic strategies. In aim 1, we will generate cerebellar organoids from five SCA4 patient-derived iPSC lines, and normal control iPSCs from individuals of the same family. These iPSC lines are already established in our laboratory. In aim 2, we will investigate PC viability, NII protein composition, proteostasis pathways, RNA gain-of-function and cell-type-specific dysregulated pathways by single nucleus RNA sequencing. In addition, we will study potential therapeutic targets by lentiviral knockdown and single nucleus RNA sequencing. SCA4 patient iPSCs express overabundant STAU1 and ATXN2. We will evaluate how lowering the abundance of these proteins modifies the PC molecular phenotype. Together, these experiments will establish a model to greatly enhance the understanding of human PC neurodegeneration, the pathological mechanisms of SCA4 and possible avenues of treatment.

Intrinsic and extrinsic mechanisms underlying trigeminal nerve deficits in familial dysautonomia

PROJECT SUMMARY Rare diseases impose a significant burden on the US healthcare system, accounting for nearly half of all expenditures for their treatment. This statistic alone supports the need to invest in research to develop therapeutic interventions for rare diseases since the economic benefit outweighs the continued expense of financial resources. Familial dysautonomia (FD) is a rare, hereditary disease that arises from a splice site mutation in Elongator acetyltransferase complex subunit 1 (ELP1) and impacts the nervous system. To date, FD patients continue to face life-threatening complications involving basic involuntary functions like swallowing and somatosensation because there is no cure for this ultimately fatal neuropathy. FD patients exhibit symptoms due to defects in their somatosensory trigeminal nerves, whose cell bodies reside in the trigeminal ganglion (TG) and are derived from neural crest and placode cells. Recent studies from our lab using an FD mouse model (Elp1 deleted from neural crest cells) revealed TG axon outgrowth and target tissue innervation deficits, recapitulating phenotypes observed in FD patients. However, the mechanisms by which Elp1 mediates normal TG development, and how this goes awry in FD, remain largely elusive. To gain insight into Elp1 function, we performed mass spectrometry to evaluate the TG proteome of normal and FD mouse embryos. Our results uncovered statistically significant increases in extracellular matrix (ECM) and ECM binding proteins, pointing to altered TG biomechanical properties and, more broadly, changes in mechanotransduction, the process by which cells translate extrinsic cues into intrinsic signaling pathways that modulate gene expression. Importantly, proper axon outgrowth relies upon mechanotransduction as growth cones on axons sense and respond to their environment. In the head, this environment consists of ECM and cranial mesenchyme cells, but the impact of Elp1 loss from the latter is not known, including the potential for altered tissue biomechanics that could influence TG axon outgrowth. We hypothesize that loss of Elp1 induces changes in the biomechanical properties of both the TG/nerves and ECM/cranial mesenchyme, modifying mechanotransduction and leading to TG defects in FD, which we will interrogate in the following Specific Aims: 1) define the biomechanical properties of the TG/nerves and ECM/cranial mesenchyme and 2) determine the role of cranial mesenchyme Elp1 in mediating proper TG axon outgrowth. Our innovative research proposal takes a systems-level, multidisciplinary approach involving embryology, biomechanics, and high-resolution microscopy, with the goal of integrating molecular, cellular, and tissue data. These results will significantly advance our knowledge of the molecular mechanisms underscoring TG development and, collectively, inform treatment strategies for birth defects or disorders like FD with TG dysfunction, as well as nerve repair and/or regeneration after injury or disease.

Optimizing gamma-delta T cell receptor-mediated signaling to improve cancer immunotherapy

PROJECT SUMMARY The recent development of T cell-based cancer immunotherapies, including checkpoint blockade (anti-PD-1, anti-CTLA-4 and others) or adoptive cell therapy (ACT) using modified patient T cells, has led to improved patient outcomes for a variety of cancers. However, durable responses are observed in only a fraction of patients. Further progress can be made by studying and targeting different T cell subpopulations, such as the gd T cells which are known to possess antitumor activities. Further, gd T cells are mostly independent of MHC-restriction, unconstrained by neoantigen burden, preferential homing to peripheral tissues and possess unique properties of T cells as well as natural killer cells making them an extremely attractive cancer immunotherapy target. One way of gd T cell activation involves the gd T cell receptor (gdTCR)-CD3 signaling pathway. gd T cell recognition of antigen by the gdTCR and the resulting proximal signaling through surrounding CD3 subunits are key steps of gd T cell activation. Even though the individual components of the gdTCR-CD3 and abTCR-CD3 complexes remain the same except for the TCRs, the complete gdTCR-CD3 complex extracellular structure is unknown. Identification of the specific extracellular interactions between the gdTCR and CD3 subunits could offer precise guidance for the development of immunotherapeutic strategies that modulate gdT cell immunity by targeting signaling through the gdTCR-CD3 complex. Our previous data showed that mutating residues in the constant domain of the abTCR resulted in altered ab T cell cytokine responses. Based on this data, our hypothesis is that gdTCR-CD3 signaling can also be modulated by targeting specific regions of the gdTCR by mutagenesis to improve gd T cell antitumor activities. To test our hypothesis, in Aim 1, we will use a novel photo-crosslinking and computational docking methodology to solve the complete extracellular structure of a gdTCR-CD3 complex. Further, we will use an in silico structure-based TCR design approach to identify gdTCR mutants that enhance signaling. In Aim 2, we will use an in vitro retroviral TCR display method using degenerate primers to create gdTCR mutant libraries at specific gdTCR sites such as Cg helix 3 and connecting peptide (CP) regions. In both instances, identified mutants will be tested for improved functionalities in an MHC-independent gd TCR (G115 Vg9Vd2 TCR) using in vitro cytokine and tumor-killing assays. Overall, the newly identified enhanced gd T cell clones could potentially lead to a new wave of effective cancer immunotherapy strategy by leaning into the largely untapped potential of gd T cells.

Implementing a New Paradigm for Antifungal Drug Development

About 30% of the drugs currently in clinical use function through covalent modification of their target. Yet, until recently, none of these covalent drugs were specifically designed to utilize this irreversible mode of action. It is our hypothesis that the production of a new class of covalent inactivators, designed to selectively modify new drug targets, will lead to novel agents with efficacy against both native and drug-resistant pathogenic fungal species. Because of their novelty these agents will also offer a greater opportunity to bypass the existing mechanisms of drug resistance. Pathogenic fungal infections remain among the leading causes of human mortality, and this threat is rising due to the increasing prevalence of drug- resistance strains and the paucity of effective antifungal drugs against the more virulent fungal species. Our proposed new drug target is an enzyme that plays a critical role in a uniquely microbial pathway that is essential for the survival of fungal organisms. To test our hypothesis and achieve the goals of this project we plan to complete the following specific aims during the initial R21 phase of this project: (1) Optimization of the potency of novel enzyme inactivators. Our goals here are to use our strong preliminary results to address critical barriers that must be overcome to convert potent enzyme inactivators into advanced drug candidates, thereby achieving higher target selectivity and increasing compound reactivity once bound to the target; (2) Enhance the antifungal capability of these enzyme inactivators. Our strategy for this aim is focused on the incorporation of conjugate partners into this new class of covalent inactivators, enabling them to potentially utilize the existing nutrient uptake systems to achieve toxic levels in Candida species; (3) Examine the target selectivity of our new antifungal agents. Results from fungal growth inhibition and fungal killing assays will be used to evaluate and rank the efficacy of our compounds against both wild-type and drug-resistant Candida strains. Specific milestones are presented to evaluate our achievement of these initial aims. Once accomplished we will immediately proceed to the R33 phase of this project, with the aims of: (4) Pharmacological evaluation of lead candidates, though ranking the drug candidates based on their ADME, pharmacokinetic and toxicity properties; and then (5) Evaluate the efficacy of our candidates against pathogenic fungal infections. A systematic infection animal model will be utilized for candidate screening to identify the best agents against disseminated fungal infections, followed by further efficacy screening in an oral infection model. Completion of these aims will produce, refine and evaluate a new class of antifungal agents with a novel mode of action against an unexplored but essential fungal target. The agents with the most promising drug profiles will then be moved into advanced preclinical trials used to select the most effective new antifungal agents.

Enteric virus-induced innate immune responses in oral tolerance

Project Summary The human gut must constantly balance between defending against harmful microbe, including virus infections, and tolerating harmless substances, like food. One important immune process called oral tolerance helps prevent the immune system from overreacting to dietary proteins such as gluten. When this tolerance breaks down, known as loss of oral tolerance (LOT), it can lead to celiac disease, where the body mounts an immune attack against gluten. Viruses that infect the gut, known as enteric viruses, can disturb the intestinal immune homeostasis and contribute to gastrointestinal diseases. Our research has found that one such virus, the Type 1 Lang (T1L) strain of reovirus, capable of infecting human and mice, can induce LOT to gluten. We discovered that T1L triggers a type of inflammatory cell death called necroptosis in intestinal epithelial cells. This cell death sends danger signals to dendritic cells (DCs) presenting dietary antigens, including gluten to T cells. These signals appear to shift DCs from a tolerance-promoting mode to one that drives inflammation and gluten-specific TH1 responses, a hallmark of celiac disease. We believe this process begins when the virus produces a specific form of RNA called Z-RNA, which is sensed by a host protein called ZBP1, triggering necroptosis and inflammation. Our research aims to understand this pathway in detail. Aim 1 will investigate how ZBP1 detects viral Z-RNA and induces necroptosis in intestinal epithelial cells. Aim 2 will examine how this necroptosis leads to LOT and will test whether blocking or engaging the pathway can prevent or induce inflammatory dietary antigen-specific TH1 immune responses. By revealing how a common virus can break oral tolerance and trigger inflammation, this study could lead to new ways to prevent or treat autoimmune and food-related disease such as celiac disease.

Targeting subtype specification as a driver of PDAC health disparities

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is refractory to current treatment strategies due in part to adaptive mechanisms of chemoresistance. Racial health disparities also confound the treatment and care of these patients. Blacks (people with African genetic ancestry) have significantly higher incidence rates of PDAC and decreased survival times compared to Caucasians (White genetic ancestry) even after socioeconomic status and tumor stages are controlled. Therefore, it is possible different racial groups exhibit unique molecular characteristics in PDAC tumors that contribute to these health disparities. The unique molecular characteristics that distinguish PDAC tumors between racial groups exhibiting disparities have the potential to identify new therapeutic targets. In a previous study, we identified 4 distinct subtypes of PDAC (Metabolic, Progenitor-like, Proliferative, and Inflammatory) that can be distinguished using multivariate analysis of quantitative proteomic data. While these PDAC subtypes are predictive of therapeutic response, this has not yet been analyzed in disparity factor balanced studies. We have examined the proteomes of primary PDAC tumors using quantitative mass spectrometry and identified unique protein signatures for Blacks and Whites. PDAC tumors from Black patients display features consistent with the Inflammatory subtype of PDAC, which is characterized by an inflamed microenvironment expressing complement proteins that can promote resistance to chemotherapy. Therefore, it is possible that race influences subtype and Blacks could preferentially develop the more aggressive and treatment refractory Inflammatory subtype. Strategies are needed to modulate subtype to improve response to chemotherapy. Toward this goal, our proteomic analysis identified polycomb repressor complex 1 (PRC1) protein RNF2 as being upregulated in PDACs from Blacks compared to Whites. We have also discovered that RNF2 regulates mRNA expression of the PDAC subtype specification factor GATA6 and inhibiting RNF2 promotes a molecular shift toward the more chemosensitive Classical subtype of PDAC. Therapeutic targeting can be achieved with Tazemetostat that inhibits the upstream PRC2 to prevent RNF2 binding the GATA6 promoter leading to its increased expression. Additionally, the Inflammatory subtype characterized by innate immune complement protein activation could be targeted with another FDA approved drug, Avacopan, which has not previously been studied in PDAC. Therefore, the Specific Aims of this proposal are designed to: 1) Evaluate the extent to which Tazemetostat treatment impacts chemotherapy-induced subtype plasticity in patient derived organoids; and 2) To determine the extent to which strategies targeting pathways associated with PDAC disparities affect progression and subtype characteristics in vivo. The successful completion of these aims has the potential to be moved quickly into phase I clinical trials since both Tazemetostat and Avacopan are FDA approved drugs. Furthermore, if successful, this project has the potential to mitigate health disparities in PDAC and broadly improve patient outcomes by implementing new precision interventions. The mouse models we propose faithfully recapitulate pancreatic cancer's clinical syndrome, histopathology and molecular properties, including the often-unique features of the stromal and immune responses that constitute the complex desmoplasia of this disease, which cannot be addressed using in vitro model systems

Spatial Mapping to Detail the Role of Biomolecules in Governing Biofilm Organization and Resiliency to Stress in Pseudomonas aeruginosa Biofilms

PROJECT SUMMARY The bacterium Pseudomonas aeruginosa is a leading cause of hospital acquired infections, exhibiting substantial antibiotic tolerance due to growth in biofilms. Our previous work shows how biofilm fitness is increased by alkyl quinolones (AQs), a class of molecules produced by the Pseudomonas Quinolone Signal (PQS) pathway of Pseudomonas aeruginosa. AQs form aggregates that spatially limit regions of cell death and reduce overall cell death in biofilms. Spatial studies build on ”what” molecules are doing by revealing when, where, and with whom they are found. Others have shown that AQs transiently bind amyloids and our preliminary results find that amyloid localization is shifted in the absence of AQs. However, the spatial relationships of these molecules have not been investigated. Our research combines multiple spatial analytical techniques, such as fluorescence microscopy, polarized light microscopy, confocal Raman microscopy to assemble detailed maps of AQ and amyloid localization during biofilm development. Using transgenic strains we will also determine amyloid distribution as a function of AQ abundance. This work will build on previous findings that AQ concentrations are able to shift locally in response to stress. We hypothesize that this can impact the localization of amyloids and allow biofilms to respond locally to stress, shielding the greater biofilm from damage. We will map biomolecular distribution of entire colony biofilms in response to stress to determine if local responses have the ability to shield more distal regions of the biofilm. The capacity of spatial biomolecular organization to increase bacterial resilience and infection virulence is an understudied area that has the potential to bring to light to novel targets for therapeutics to fight biofilm infections.

AI-guided structural biology of Cav1.2

Project Summary/Abstract The L-type calcium channel Cav1.2 plays a critical role in excitation-contraction coupling in the heart. Its calcium flux generates the plateau phase of the cardiac action potential and results in the calcium-induced calcium release needed to trigger cardiac contractions. Cav1.2 is a multi-subunit protein consisting of a large, transmembrane 1 subunit and smaller, auxiliary subunits important for trafficking and channel regulation. Recent cryogenic electron microscopy (cryo-EM) experiments have revealed much of the three-dimensional structure of Cav1.2’s core domains, though the final 571 residues of the 1 subunit’s intracellular C-terminal domain (CTD) have not yet been resolved despite key regulatory roles in channel function. This domain has been shown to be important for Cav1.2’s regulation by calcium/calmodulin and has an important role in cross- talk between Cav1.2 and the sympathetic nervous system, amongst other cell signaling pathways. In this proposal, I will use insights from artificial intelligence to develop a platform for CTD structural biology, then validate that platform by measuring its ability to form protein-protein interactions with known binding partners of Cav1.2, including calcium/calmodulin and an autoregulatory distal C-terminal fragment. If successful, I will also attempt crystallization of the CTD in complex with several binding partners. Together these data will provide the starting point for future structural biology projects on Cav1.2 regulation and protein-protein interactions.

Programming Offspring Metabolism: The Role of Milk Extracellular Vesicles in Fat Development

SUMMARY Obesity is a global health crisis, contributing significantly to the prevalence of metabolic disorders, cardiovascular diseases, and various chronic conditions. A growing body of evidence suggests that maternal obesity during pregnancy and lactation can predispose offspring to obesity and metabolic dysfunction later in life. However, the mechanisms by which maternal obesity programs these adverse outcomes in offspring remain poorly understood. Breast milk is not only a source of essential nutrients but also contains bioactive components, including extracellular vesicles (EVs), which play crucial roles in cellular communication and development. Recent studies have shown that EVs can survive digestion and enter the infant’s circulation, influencing immune and metabolic development. Despite the established link between maternal obesity and altered breast milk composition, no study has investigated the role of milk-derived EVs (mEVs) in programming offspring fat development and metabolism. Understanding this novel pathway could revolutionize our approach to preventing intergenerational transmission of obesity. Our preliminary studies using a mouse model of maternal high-fat diet-induced obesity revealed significant alterations in mEV biogenesis and cargo composition, including changes in specific miRNAs. Oral administration of mEVs from obese dams to neonatal mice increased adiposity and impaired lipid metabolism, indicating that mEVs are crucial in modulating fat development and metabolic pathways in offspring. Several key miRNAs found in mouse mEVs are conserved in human milk EVs, highlighting the potential translational relevance of our findings to human health. We hypothesize that mEVs are critical mediators of maternal obesity’s programming effects on offspring metabolism and adiposity. In specific aim 1, we will use mouse models and advanced molecular techniques (miRNA sequencing, proteomics, and lipidomics) to characterize how maternal obesity affects mEV biogenesis and the composition of their bioactive cargo. We will also evaluate how maternal dietary intake, independent of obesity, influences mEV composition. Specific aim 2 will define the programming effects of mEVs on offspring energy metabolism and obesity. In addition, we will explore whether human milk EVs from lean and obese mothers exert similar programming effects on fat development and metabolism in a mouse model. This R21 application embodies a high-risk, high-reward approach to obesity research. It ventures into uncharted territory by proposing that mEVs are novel regulators of metabolic programming, a concept that has not been explored in prior studies. The potential reward is substantial: discovering a new mechanism by which maternal obesity influences offspring health could fundamentally shift our understanding of early-life metabolic programming and lead to innovative strategies for obesity prevention. If successful, this research could open a new field of study with broad implications for maternal and child health.

Autoreactive T cells in lupus

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of adaptive immune tolerance in conjunction with innate immune system hyperactivity. Autoantibodies, produced by plasma cells derived from activated B cells, form proinflammatory immune complexes. These immune complexes drive feed forward loops that sustain a systemic inflammatory environment and deposit in tissues leading to potentially fatal organ damage. B cells receive help from T cells to produce antibodies. They also contribute to disease by shaping T cell responses and secreting cytokines. Recent case reports in which SLE patients were treated with anti-CD19 CAR-T cell therapy to deplete B cells highlight the pathogenic role of B cells in lupus and their value as a therapeutic target. However, a better understanding of how autoreactive B cells interact with autoreactive T cells may reveal more targeted points of therapeutic intervention that specifically block autoreactive responses while sparing protective ones. Antigen specific interactions between CD4+ T cells and B cells are required for the development of autoimmune disease in lupus. However, whether these critical interactions occur in germinal centers, where competition for CD4+ T cell help selects high affinity B cells, or in extrafollicular responses, where B cells may avoid peripheral tolerance checkpoints, is unclear. Gene expression profiles and pathways specific to autoreactive CD4+ T cells, and how they are shaped by their interaction with autoreactive B cells, are also ill defined. CD8+ T cells, which recognize antigen presented on MHC Class I, have also been suggested to modulate the fate of autoreactive B cells. They can directly kill autoreactive B cells as a means of tolerance, and a subset of CD8+ T cells has recently been shown to have B cell helper function. Whether and how such interactions between B and CD8+ T cells enhance or suppress the development of lupus is unknown. Here, we will use genetic and in vivo proximity labeling approaches to address these knowledge gaps. In Aim 1, we will test the hypothesis that antigen specific interactions between B and CD8+ T cells promote B cell activation and autoantibody production in lupus. We will prevent B cells, but not other cells, from undergoing cognate interactions with CD8+ T cells via B cell-specific deletion of B2M, a component of the MHC Class I complex, in two lupus models. In Aim 2, will use the uLIPSTIC in vivo proximity system to label all T cells interacting with B cells in lupus models compared to wild type controls. Features specific to these autoreactive T cells will be defined by flow cytometry, scRNA Seq, and scTCR-Seq. These studies will provide valuable molecular and cellular insight into the mutual activation of B and T cells in lupus. They will set the stage for future mechanistic studies defining the role of autoreactive T cell specific genes and pathways and potentially highlight new therapeutic targets specific to autoreactive B/T interactions.

2-Deoxyglucose Therapy for Organophosphate Intoxication

Project Summary The main goal of this project is to determine the therapeutic potential of glycolysis inhibition as an adjunct to midazolam therapy in mitigating the long-term neurological effects from acute organophosphate pesticide and nerve agent (OPNA) exposure. Novel countermeasures are desperately needed for effective mitigation of morbidity and long-term effects of OPNAs. A variety of agents targeting glutamate, GABA and oxidative stress have been proposed, but glycolysis inhibitors have not been widely studied in OPNA intoxication. Dysregulated glucose metabolism plays a key role in seizures and neuronal injury following OPNA exposure. 2-Deoxyglucose (2-DG), a selective glycolysis inhibitor, has anticonvulsant and neuroprotection effects and hence can effectively mitigate acute and long-term OPNA neurotoxicity. In this project, we seek to identify the glycolysis inhibition as novel adjunct neuroprotection to midazolam therapy for OPNA exposure, with the goal of identifying 2-DG or related drugs as medical countermeasures. The glycolytic pathway represents a logical target for such intervention because glycolysis controls seizures and neuronal injury by regulating glucose utilization and activity in neurons and astrocytes in the brain. The proposed therapy is based on the hypothesis that acute OPNA neurotoxicity imparts sustained activation of the glycolysis pathway in the brain and therefore, 2- DG and selective glycolysis inhibitors prevents long-term neuronal damage neurological dysfunction. This hypothesis will be tested by using the FDA-approved (2-DG) or clinical-stage glycolytic inhibitors in two distinct OPNA models in rats: (Aim 1) To investigate the protective efficacy of 2-DG and novel glycolysis inhibitors against DFP-induced acute and long-term neuronal damage and neurological dysfunction. (Aim 2) Aim 2 (Year 2). To determine brain penetration, pilot toxicity and pharmacokinetic of 2-DG or other lead drug in naïve and DFP-exposed animals. Test drugs will be evaluated as per the NIH rigor criteria in a dose-related design in male and female rats and behavior/neuropathology will be checked for 3 months post-exposure. 2-DG and test drugs will be given starting 40-min after exposure to ONAs. Three primary outcome measures will be addressed for therapy effectiveness: (i) acute adjunct neuroprotection; (ii) chronic neuroprotectant efficacy; and (iii) prevention of neurological and behavioral deficits. The primary measures of neuroprotection include longitudinal MRI scanning, and extent of neurodegeneration, neuroinflammation, aberrant neurogenesis, and mossy fiber sprouting. Key neurological outcomes include memory deficits, depression, anxiety behavior, and neurological/motor deficits. The outcome of this project will provide “proof-of-efficacy” of a novel glycolytic therapy with FDA-approvable, repurposed drugs with promising potential to limit long-term effects of OPNAs in humans. Thus, the overall impact of the outcome is enormous for civilians, especially in developing a highly effective and safe post-exposure medical countermeasure for chemical nerve agents.

Structure-function and mechanistic studies of a specific glycosyltransferase complex in fusion-driven pediatric gliomas

Abstract Glycosylation is a co/post-translational modification involved in cell-matrix interactions, antigen-antibody interactions, tumor invasion, and cell motility. Abnormal glycosylation is a hallmark of cancer, with various glycosylation-related genes linked to glioma prognosis and tumor heterogeneity. Pediatric low-grade gliomas (pLGGs) stand as the most common childhood central nervous system tumor, accounting for 30%-40% of all CNS tumors in children. Despite its relatively low mortality rate, pLGGs are associated with devastating lifelong morbidity. The most common alteration found in 75% of tumors is the KIAA1549:BRAF fusion, causing an aberrant activation of the MAPK/ERK signaling pathway. Current treatments, such as traditional chemotherapies and targeted therapies, have limitations such as resistance, lack of specificity, toxicity and paradoxical activation of the MAPK pathway. This highlights the urgent need for novel therapeutic approaches. Investigations into KIAA1549:BRAF-driven pLGGs identified their dependency on the protein-O-mannosyl transferase (POMT) complex for survival. In contrast, BRAFV600E-mutant cells did not show dependency, suggesting the POMT complex as a vulnerability and promising target in KIAA1549:BRAF-driven pLGGs. Therefore, our goal is to characterize the POMT complex structurally and biochemically and study its roles in KIAA1549:BRAF-driven pLGGs. In this proposal, we aim to 1) determine the high-resolution structures of the complex in its unbound, substrate-bound, and inhibitor-bound forms and 2) elucidate the POMT complex mechanisms in KIAA1549:BRAF-driven pLGGs. We will define the critical functional domains, active sites, interaction interfaces and translational modifications crucial for enzymatic activity using cryo-EM techniques, mutagenesis, and functional studies. To study biological pathways and molecular events modulated by the POMT complex, we will implement global proteomics and transcriptomics analysis in well-characterized disease models. In parallel, we will assess the effect of the POMT complex on the MAPK/ERK signaling pathway. This study will guide the structure-based design of probes and drugs targeting the POMT complex and will unveil glycosylation-mediated oncogenesis in pediatric gliomas. It will aid in the development of new targeted therapies and the identification of new biomarkers for pLGGs harboring the KIAA1549:BRAF fusion. The research will be conducted in the Fischer lab at Dana-Farber Cancer Institute, which provides a collaborative and resource-rich environment. The career development plan includes training in scientific writing, mentoring, and presentation skills, as well as interdisciplinary networking with experts in structural biology and pediatric oncology. The candidate’s career goal is to establish an independent research laboratory focused on developing new therapeutic modalities for pediatric neurooncology. The training provided through this fellowship represents a critical step toward achieving this goal.

Dual mRNA Therapeutics for Liver Metastatic Uveal Melanoma

Abstract Uveal melanoma (UM) is the most common primary intraocular cancer in adults, accounting for approximately 70% of all ocular malignancies. Current treatments for primary UM include surgical tumor removal, transpupillary thermotherapy, and radiotherapy. Unfortunately, both surgical enucleation and brachytherapy have shown similar survival outcomes and carry an equivalent risk of metastasis. While the survival rate for patients with primary, non-metastatic UM is relatively high, metastatic uveal melanoma (MUM), especially when it spreads to the liver, remains universally fatal. The liver is the first site of metastasis in 80 to 90 percent of cases, and about 50 percent of UM patients develop liver metastases within 15 years of initial diagnosis. Median survival following liver metastasis is only 5 to 7 months, with an almost zero percent five-year survival rate. Currently, no available therapy significantly improves outcomes for patients with liver MUM. This R21 project addresses this urgent unmet need by developing liver-tropic mRNA therapeutics targeting two key drivers of MUM progression and metastasis: (1) constitutive activation of Gαq/11 caused by single-point mutations, and (2) loss-of-function mutations in BAP1. Both alterations occur in over 80 percent of UM patients and are associated with poor prognosis. We hypothesize that inhibition of constitutively active Gαq/11 and/or restoration of BAP1 tumor suppressor function will significantly suppress MUM progression and improve survival outcomes. Aim 1 focuses on delivering mRNA encoding a novel protein trap designed to specifically inhibit constitutively active Gαq/11 and its downstream oncogenic signaling pathways. Aim 2 seeks to restore wild-type BAP1, which is mutated or lost in approximately 84 percent of MUM cases, through liver-tropic mRNA delivery using a liver MUM model established via splenic inoculation. We will also evaluate the potential synergy between Gαq/11 inhibition and BAP1 restoration. The success of this project will not only advance our understanding of the disease mechanisms underlying MUM but also provide clinically viable strategies for treating liver metastases in uveal melanoma.

Bacterial ferrous iron sensing via the BqsRS (CarRS) two-component system

Project Summary Pseudomonas aeruginosa (Pa) is an opportunistic and increasingly antibiotic resistant Gram-negative bacterium that is one of the major causes of chronic nosocomial infections in the United States. The colonization of Pa within a host is often linked to the bioavailability of nutrients, such as iron, and Pa has multiple iron acquisition pathways that allow it to adapt readily to the variety of environments it may encounter within a human host. Pa responds to these dynamic environments commonly through the use of two-component signal transduction systems (TCSs) that are important mediators of signal transduction and allow pathogens to detect chemical and/or physical changes in the environment in order to control basic cellular processes. Previous studies have identified a biofilm and quorum sensing TCS known as BqsRS (also known as CarRS) that regulates biofilm formation and decay in Pa through the sensing of extracytoplasmic Fe2+ and Ca2+. Among its targets, the BqsRS TCS is known to regulate rhlAB and rhlC, critical genes for rhamnolipid production and biofilm formation that are also known to be connected to iron homeostasis and antibiotic resistance. Moreover, the deletion of either bqsR or bqsS in PAO1 results in a significant increase in biofilm formation but reduced biofilm dispersion, the latter of which is important for downstream infections. These observations highlight the importance of the BqsRS TCS to Pa virulence, but there is a foundational lack of understanding regarding the structure, the selectivity, and the mechanism of this system. The ultimate goal of this proposal is to generate a mechanistic and functional understanding of BqsRS at atomic, molecular, and organismal levels in order to exploit this system as a means of reducing or stemming the virulence of opportunistic pathogens such as Pa. The objectives of this exploratory grant are to determine the structural and molecular characteristics of BqsRS, to define how these properties govern BqsRS metal selectivity and function, and to examine a new role of the BqsRS system in regulating the Feo system in P. aeruginosa. Ultimately, the accomplishment of this exploratory grant will deliver fundamental mechanistic insight into a critical metal-sensing TCS and lay the groundwork for future studies that may be designed to target this system and its homologs for additional bacterial exploits.

Noninvasive Neuromodulation to Improve Hand Motor Function in Multiple Sclerosis

Project Summary/Abstract Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating, and degenerative disease that affects nearly one million Americans. Although more than 75% of persons with MS (PwMS) experience hand motor impairments that reduce independence and quality of life, current treatments primarily aim to slow disease progression through pharmacological approaches and rehabilitation and often do not improve motor function. Recent evidence shows that reduced corticospinal transmission is strongly associated with motor impairment severity in PwMS, highlighting the need for targeted strategies to strengthen residual corticospinal pathways. Therefore, this project aims to evaluate the therapeutic potential of paired corticospinal-motoneuronal stimulation (PCMS) in improving hand dexterity in PwMS. PCMS, a noninvasive mechanism-driven neuromodulation approach, enhances corticospinal transmission by producing long-term potentiation-like effects at the corticospinal-motoneuronal synapse by precisely pairing transcranial magnetic stimulation (TMS) with peripheral nerve stimulation (PNS). This project first aims to examine the effects of a single PCMS session on corticospinal transmission and hand motor function in PwMS. Using a randomized, crossover design, 25 PwMS will complete two sessions: (1) PCMS and (2) sham-PCMS. Each session will deliver 180 paired TMS-PNS stimuli over 30 minutes. The primary outcome is performance on the 9-Hole Peg Test (9HPT). Secondary outcomes include pinch grip force, maximal voluntary contraction (MVC), MEP amplitude and latency, F-wave parameters, and M- max amplitude. It is hypothesized that PCMS will enhance corticospinal transmission and improve hand motor performance compared to sham stimulation. Second, this project will examine the effects of PCMS combined with hand motor training in PwMS. Forty-eight PwMS will be randomized to receive either PCMS or sham-PCMS combined with motor training over 10 sessions in 3–4 weeks. Outcomes will be assessed at baseline, post- intervention, and one-month follow-up. It is hypothesized that PCMS participants receiving PCMS with motor training to show greater functional gains than those receiving sham-PCMS with motor training and the functional gains will be better maintained in the PCMS with motor training group at follow-up. This project is the first to apply PCMS in PwMS, leveraging a noninvasive neuromodulation strategy to specifically enhance corticospinal output for improving manual dexterity. Findings will establish proof-of-concept for this intervention in PwMS and guide future studies optimizing stimulation protocols and evaluating clinical efficacy on a larger scale. Ultimately, this work may lead to a new therapeutic approach to improve dexterity, independence, and quality of life for people living with MS.

Glycoengineering core a(1,3)-fucose motifs to enhance HIV-1 envelope vaccine immunogenicity

Project Summary The HIV-1 envelope glycoprotein (Env) is the sole target of neutralizing antibodies (NAbs). We previously developed a vaccine platform integrating three innovations: (1) the uncleaved prefusion-optimized (UFO) trimer design to stabilize Env; (2) multilayered single-component self-assembling protein nanoparticles (1c-SApNPs) for multivalent trimer display; and (3) enzymatic trimming of oligomannose glycans on CHO cell-produced Env immunogens. Glycan trimming substantially improved Env immunogenicity by enhancing tier 2 NAb elicitation, reducing off-target responses to immunodominant glycan sites, and increasing responder rates. These vaccine candidates are now in phase 1 clinical trials (NCT06541093; NCT06905275). Building on this foundation, we propose a novel strategy to enhance immunogenicity by incorporating core α(1,3)-fucose into HIV-1 Env. Core α(1,3)-fucose, a key allergenic epitope in many plant and insect glycoproteins, is highly immunogenic in humans and other mammals. Our central hypothesis is that the targeted introduction of core α(1,3)-fucose will convert the glycan shield from an immune-evasive barrier into an immunogenic trigger that promotes NAb induction. Glycoengineered cell lines expressing α(1,3)-fucose will enable production of highly immunogenic Env vaccines suitable for preclinical and clinical testing. Importantly, particulate display of these Env trimers on 1c-SApNPs can suppress IgE-mediated allergic pathways by inducing high-affinity protective IgGs, ensuring vaccine safety. Aim 1 will focus on producing core α(1,3)-fucosylated HIV-1 Env immunogens. We will begin by developing a transient insect cell expression system using BTI-TN-5B1-4 (“High Five” or Hi5) cells to produce Env with short paucimannose glycans bearing native α(1,3)-fucose. To further enhance α(1,3)-fucosylation, we will co-express exogenous core α(1,3)-fucosyltransferases in insect and CHO cells. We will validate glycan profiles and characterize the biochemical, biophysical, structural, and antigenic properties of the resulting immunogens. Aim 2 will assess the immunogenicity of these glycoengineered HIV-1 Env immunogens. Using our previously established glycan-trimmed Env immunogens as benchmarks, we will immunize mice, rabbits, and nonhuman primates (NHPs). Mice will be used for early-stage immunogen and adjuvant screening; rabbits to evaluate glycan hole-targeting NAb responses; and key vaccine formulations will advance to NHP studies. We will assess autologous and heterologous tier 2 NAb responses and vaccine responder rates. Aim 3 will elucidate the functional, structural, repertoire, and mechanistic basis of vaccine-induced immunity. We will isolate NAbs via Env-specific single-cell sorting and antibody cloning, map epitopes by electron microscopy (EM) and X-ray crystallography, perform next-generation sequencing (NGS) of B-cell repertoires, and trace NAb lineages. Finally, we will investigate antigen trafficking, retention, presentation, and germinal center (GC) reactions in lymph nodes. Together, these studies will define a new class of glycoengineered HIV-1 vaccines and establish core α(1,3)-fucose as a novel immunomodulatory tool to overcome glycan shield-mediated immune evasion.

Computational Mechanisms of Predictive Processing in Brains and Machines

Predictive processing offers a unifying view of neural computation, proposing that brains continuously anticipate sensory input and update internal models based on prediction errors. In this talk, I will present converging evidence for the computational mechanisms underlying this framework across human neuroscience and deep neural networks. I will begin with recent work showing that large-scale distributed prediction-error encoding in the human brain directly predicts how sensory representations reorganize through predictive learning. I will then turn to PredNet, a popular predictive coding inspired deep network that has been widely used to model real-world biological vision systems. Using dynamic stimuli generated with our Spatiotemporal Style Transfer algorithm, we demonstrate that PredNet relies primarily on low-level spatiotemporal structure and remains insensitive to high-level content, revealing limits in its generalization capacity. Finally, I will discuss new recurrent vision models that integrate top-down feedback connections with intrinsic neural variability, uncovering a dual mechanism for robust sensory coding in which neural variability decorrelates unit responses, while top-down feedback stabilizes network dynamics. Together, these results outline how prediction error signaling and top-down feedback pathways shape adaptive sensory processing in biological and artificial systems.

Organization of thalamic networks and mechanisms of dysfunction in schizophrenia and autism

Thalamic networks, at the core of thalamocortical and thalamosubcortical communications, underlie processes of perception, attention, memory, emotions, and the sleep-wake cycle, and are disrupted in mental disorders, including schizophrenia and autism. However, the underlying mechanisms of pathology are unknown. I will present novel evidence on key organizational principles, structural, and molecular features of thalamocortical networks, as well as critical thalamic pathway interactions that are likely affected in disorders. This data can facilitate modeling typical and abnormal brain function and can provide the foundation to understand heterogeneous disruption of these networks in sleep disorders, attention deficits, and cognitive and affective impairments in schizophrenia and autism, with important implications for the design of targeted therapeutic interventions

How the presynapse forms and functions”

Nervous system function relies on the polarized architecture of neurons, established by directional transport of pre- and postsynaptic cargoes. While delivery of postsynaptic components depends on the secretory pathway, the identity of the membrane compartment(s) that supply presynaptic active zone (AZ) and synaptic vesicle (SV) proteins is largely unknown. I will discuss our recent advances in our understanding of how key components of the presynaptic machinery for neurotransmitter release are transported and assembled focussing on our studies in genome-engineered human induced pluripotent stem cell-derived neurons. Specifically, I will focus on the composition and cell biological identity of the axonal transport vesicles that shuttle key components of neurotransmission to nascent synapses and on machinery for axonal transport and its control by signaling lipids. Our studies identify a crucial mechanism mediating the delivery of SV and active zone proteins to developing synapses and reveal connections to neurological disorders. In the second part of my talk, I will discuss how exocytosis and endocytosis are coupled to maintain presynaptic membrane homeostasis. I will present unpublished data regarding the role of membrane tension in the coupling of exocytosis and endocytosis at synapses. We have identified an endocytic BAR domain protein that is capable of sensing alterations in membrane tension caused by the exocytotic fusion of SVs to initiate compensatory endocytosis to restore plasma membrane area. Interference with this mechanism results in defects in the coupling of presynaptic exocytosis and SV recycling at human synapses.

Astrocytes release glutamate by regulated exocytosis in health and disease

Astrocytes release glutamate by regulated exocytosis in health and disease Vladimir Parpura, International Translational Neuroscience Research Institute, Zhejiang Chinese Medical University, Hangzhou, P.R. China Parpura will present you with the evidence that astrocytes, a subtype of glial cells in the brain, can exocytotically release the neurotransmitter glutamate and how this release is regulated. Spatiotemporal characteristic of vesicular fusion that underlie glutamate release in astrocytes will be discussed. He will also present data on a translational project in which this release pathway can be targeted for the treatment of glioblastoma, the deadliest brain cancer.

The cellular phase of Alzheimer’s Disease and the path towards therapies

LRRK2 – a master regulator of neurodegeneration: acting on multiple systems including neuroinflammatory signaling, vesicular trafficking, and cell death pathways

Vision for perception versus vision for action: dissociable contributions of visual sensory drives from primary visual cortex and superior colliculus neurons to orienting behaviors

The primary visual cortex (V1) directly projects to the superior colliculus (SC) and is believed to provide sensory drive for eye movements. Consistent with this, a majority of saccade-related SC neurons also exhibit short-latency, stimulus-driven visual responses, which are additionally feature-tuned. However, direct neurophysiological comparisons of the visual response properties of the two anatomically-connected brain areas are surprisingly lacking, especially with respect to active looking behaviors. I will describe a series of experiments characterizing visual response properties in primate V1 and SC neurons, exploring feature dimensions like visual field location, spatial frequency, orientation, contrast, and luminance polarity. The results suggest a substantial, qualitative reformatting of SC visual responses when compared to V1. For example, SC visual response latencies are actively delayed, independent of individual neuron tuning preferences, as a function of increasing spatial frequency, and this phenomenon is directly correlated with saccadic reaction times. Such “coarse-to-fine” rank ordering of SC visual response latencies as a function of spatial frequency is much weaker in V1, suggesting a dissociation of V1 responses from saccade timing. Consistent with this, when we next explored trial-by-trial correlations of individual neurons’ visual response strengths and visual response latencies with saccadic reaction times, we found that most SC neurons exhibited, on a trial-by-trial basis, stronger and earlier visual responses for faster saccadic reaction times. Moreover, these correlations were substantially higher for visual-motor neurons in the intermediate and deep layers than for more superficial visual-only neurons. No such correlations existed systematically in V1. Thus, visual responses in SC and V1 serve fundamentally different roles in active vision: V1 jumpstarts sensing and image analysis, but SC jumpstarts moving. I will finish by demonstrating, using V1 reversible inactivation, that, despite reformatting of signals from V1 to the brainstem, V1 is still a necessary gateway for visually-driven oculomotor responses to occur, even for the most reflexive of eye movement phenomena. This is a fundamental difference from rodent studies demonstrating clear V1-independent processing in afferent visual pathways bypassing the geniculostriate one, and it demonstrates the importance of multi-species comparisons in the study of oculomotor control.

Memory formation in hippocampal microcircuit

The centre of memory is the medial temporal lobe (MTL) and especially the hippocampus. In our research, a more flexible brain-inspired computational microcircuit of the CA1 region of the mammalian hippocampus was upgraded and used to examine how information retrieval could be affected under different conditions. Six models (1-6) were created by modulating different excitatory and inhibitory pathways. The results showed that the increase in the strength of the feedforward excitation was the most effective way to recall memories. In other words, that allows the system to access stored memories more accurately.

Neurobiological Pathways to Tau-dependent Pathology: Perspectives from flies to humans

Sensory cognition

This webinar features presentations from SueYeon Chung (New York University) and Srinivas Turaga (HHMI Janelia Research Campus) on theoretical and computational approaches to sensory cognition. Chung introduced a “neural manifold” framework to capture how high-dimensional neural activity is structured into meaningful manifolds reflecting object representations. She demonstrated that manifold geometry—shaped by radius, dimensionality, and correlations—directly governs a population’s capacity for classifying or separating stimuli under nuisance variations. Applying these ideas as a data analysis tool, she showed how measuring object-manifold geometry can explain transformations along the ventral visual stream and suggested that manifold principles also yield better self-supervised neural network models resembling mammalian visual cortex. Turaga described simulating the entire fruit fly visual pathway using its connectome, modeling 64 key cell types in the optic lobe. His team’s systematic approach—combining sparse connectivity from electron microscopy with simple dynamical parameters—recapitulated known motion-selective responses and produced novel testable predictions. Together, these studies underscore the power of combining connectomic detail, task objectives, and geometric theories to unravel neural computations bridging from stimuli to cognitive functions.

Learning representations of specifics and generalities over time

There is a fundamental tension between storing discrete traces of individual experiences, which allows recall of particular moments in our past without interference, and extracting regularities across these experiences, which supports generalization and prediction in similar situations in the future. One influential proposal for how the brain resolves this tension is that it separates the processes anatomically into Complementary Learning Systems, with the hippocampus rapidly encoding individual episodes and the neocortex slowly extracting regularities over days, months, and years. But this does not explain our ability to learn and generalize from new regularities in our environment quickly, often within minutes. We have put forward a neural network model of the hippocampus that suggests that the hippocampus itself may contain complementary learning systems, with one pathway specializing in the rapid learning of regularities and a separate pathway handling the region’s classic episodic memory functions. This proposal has broad implications for how we learn and represent novel information of specific and generalized types, which we test across statistical learning, inference, and category learning paradigms. We also explore how this system interacts with slower-learning neocortical memory systems, with empirical and modeling investigations into how the hippocampus shapes neocortical representations during sleep. Together, the work helps us understand how structured information in our environment is initially encoded and how it then transforms over time.

How are the epileptogenesis clocks ticking?

The epileptogenesis process is associated with large-scale changes in gene expression, which contribute to the remodelling of brain networks permanently altering excitability. About 80% of the protein coding genes are under the influence of the circadian rhythms. These are 24-hour endogenous rhythms that determine a large number of daily changes in physiology and behavior in our bodies. In the brain, the master clock regulates a large number of pathways that are important during epileptogenesis and established-epilepsy, such as neurotransmission, synaptic homeostasis, inflammation, blood-brain barrier among others. In-depth mapping of the molecular basis of circadian timing in the brain is key for a complete understanding of the cellular and molecular events connecting genes to phenotypes.

Blood-brain barrier dysfunction in epilepsy: Time for translation

The neurovascular unit (NVU) consists of cerebral blood vessels, neurons, astrocytes, microglia, and pericytes. It plays a vital role in regulating blood flow and ensuring the proper functioning of neural circuits. Among other, this is made possible by the blood-brain barrier (BBB), which acts as both a physical and functional barrier. Previous studies have shown that dysfunction of the BBB is common in most neurological disorders and is associated with neural dysfunction. Our studies have demonstrated that BBB dysfunction results in the transformation of astrocytes through transforming growth factor beta (TGFβ) signaling. This leads to activation of the innate neuroinflammatory system, changes in the extracellular matrix, and pathological plasticity. These changes ultimately result in dysfunction of the cortical circuit, lower seizure threshold, and spontaneous seizures. Blocking TGFβ signaling and its associated pro-inflammatory pathway can prevent this cascade of events, reduces neuroinflammation, repairs BBB dysfunction, and prevents post-injury epilepsy, as shown in experimental rodents. To further understand and assess BBB integrity in human epilepsy, we developed a novel imaging technique that quantitatively measures BBB permeability. Our findings have confirmed that BBB dysfunction is common in patients with drug-resistant epilepsy and can assist in identifying the ictal-onset zone prior to surgery. Current clinical studies are ongoing to explore the potential of targeting BBB dysfunction as a novel treatment approach and investigate its role in drug resistance, the spread of seizures, and comorbidities associated with epilepsy.

Astrocyte reprogramming / activation and brain homeostasis

Astrocytes are multifunctional glial cells, implicated in neurogenesis and synaptogenesis, supporting and fine-tuning neuronal activity and maintaining brain homeostasis by controlling blood-brain barrier permeability. During the last years a number of studies have shown that astrocytes can also be converted into neurons if they force-express neurogenic transcription factors or miRNAs. Direct astrocytic reprogramming to induced-neurons (iNs) is a powerful approach for manipulating cell fate, as it takes advantage of the intrinsic neural stem cell (NSC) potential of brain resident reactive astrocytes. To this end, astrocytic cell fate conversion to iNs has been well-established in vitro and in vivo using combinations of transcription factors (TFs) or chemical cocktails. Challenging the expression of lineage-specific TFs is accompanied by changes in the expression of miRNAs, that post-transcriptionally modulate high numbers of neurogenesis-promoting factors and have therefore been introduced, supplementary or alternatively to TFs, to instruct direct neuronal reprogramming. The neurogenic miRNA miR-124 has been employed in direct reprogramming protocols supplementary to neurogenic TFs and other miRNAs to enhance direct neurogenic conversion by suppressing multiple non-neuronal targets. In our group we aimed to investigate whether miR-124 is sufficient to drive direct reprogramming of astrocytes to induced-neurons (iNs) on its own both in vitro and in vivo and elucidate its independent mechanism of reprogramming action. Our in vitro data indicate that miR-124 is a potent driver of the reprogramming switch of astrocytes towards an immature neuronal fate. Elucidation of the molecular pathways being triggered by miR-124 by RNA-seq analysis revealed that miR-124 is sufficient to instruct reprogramming of cortical astrocytes to immature induced-neurons (iNs) in vitro by down-regulating genes with important regulatory roles in astrocytic function. Among these, the RNA binding protein Zfp36l1, implicated in ARE-mediated mRNA decay, was found to be a direct target of miR-124, that be its turn targets neuronal-specific proteins participating in cortical development, which get de-repressed in miR-124-iNs. Furthermore, miR-124 is potent to guide direct neuronal reprogramming of reactive astrocytes to iNs of cortical identity following cortical trauma, a novel finding confirming its robust reprogramming action within the cortical microenvironment under neuroinflammatory conditions. In parallel to their reprogramming properties, astrocytes also participate in the maintenance of blood-brain barrier integrity, which ensures the physiological functioning of the central nervous system and gets affected contributing to the pathology of several neurodegenerative diseases. To study in real time the dynamic physical interactions of astrocytes with brain vasculature under homeostatic and pathological conditions, we performed 2-photon brain intravital imaging in a mouse model of systemic neuroinflammation, known to trigger astrogliosis and microgliosis and to evoke changes in astrocytic contact with brain vasculature. Our in vivo findings indicate that following neuroinflammation the endfeet of activated perivascular astrocytes lose their close proximity and physiological cross-talk with vasculature, however this event is at compensated by the cross-talk of astrocytes with activated microglia, safeguarding blood vessel coverage and maintenance of blood-brain integrity.

Connectome-based models of neurodegenerative disease

Neurodegenerative diseases involve accumulation of aberrant proteins in the brain, leading to brain damage and progressive cognitive and behavioral dysfunction. Many gaps exist in our understanding of how these diseases initiate and how they progress through the brain. However, evidence has accumulated supporting the hypothesis that aberrant proteins can be transported using the brain’s intrinsic network architecture — in other words, using the brain’s natural communication pathways. This theory forms the basis of connectome-based computational models, which combine real human data and theoretical disease mechanisms to simulate the progression of neurodegenerative diseases through the brain. In this talk, I will first review work leading to the development of connectome-based models, and work from my lab and others that have used these models to test hypothetical modes of disease progression. Second, I will discuss the future and potential of connectome-based models to achieve clinically useful individual-level predictions, as well as to generate novel biological insights into disease progression. Along the way, I will highlight recent work by my lab and others that is already moving the needle toward these lofty goals.

Neuroinflammation in Epilepsy: what have we learned from human brain tissue specimens ?

Epileptogenesis is a gradual and dynamic process leading to difficult-to-treat seizures. Several cellular, molecular, and pathophysiologic mechanisms, including the activation of inflammatory processes.  The use of human brain tissue represents a crucial strategy to advance our understanding of the underlying neuropathology and the molecular and cellular basis of epilepsy and related cognitive and behavioral comorbidities,  The mounting evidence obtained during the past decade has emphasized the critical role of inflammation  in the pathophysiological processes implicated in a large spectrum of genetic and acquired forms of  focal epilepsies. Dissecting the cellular and molecular mediators of  the pathological immune responses and their convergent and divergent mechanisms, is a major requisite for delineating their role in the establishment of epileptogenic networks. The role of small regulatory molecules involved in the regulation of  specific pro- and anti-inflammatory pathways  and the crosstalk between neuroinflammation and oxidative stress will be addressed.    The observations supporting the activation of both innate and adaptive immune responses in human focal epilepsy will be discussed and elaborated, highlighting specific inflammatory pathways as potential targets for antiepileptic, disease-modifying therapeutic strategies.

The role of CNS microglia in health and disease

Microglia are the resident CNS macrophages of the brain parenchyma. They have many and opposing roles in health and disease, ranging from inflammatory to anti-inflammatory and protective functions, depending on the developmental stage and the disease context. In Multiple Sclerosis, microglia are involved to important hallmarks of the disease, such as inflammation, demyelination, axonal damage and remyelination, however the exact mechanisms controlling their transformation towards a protective or devastating phenotype during the disease progression remains largely unknown until now. We wish to understand how brain microglia respond to demyelinating insults and how their behaviour changes in recovery. To do so we developed a novel histopathological analysis approach in 3D and a cell-based analysis tool that when applied in the cuprizone model of demyelination revealed region- and disease- dependent changes in microglial dynamics in the brain grey matter during demyelination and remyelination. We now use similar approaches with the aim to unravel sensitive changes in microglial dynamics during neuroinflammation in the EAE model. Furthermore, we employ constitutive knockout and tamoxifen-inducible gene-targeting approaches, immunological techniques, genetics and bioinformatics and currently seek to clarify the specific role of the brain resident microglial NF-κB molecular pathway versus other tissue macrophages in EAE.

How fly neurons compute the direction of visual motion

Detecting the direction of image motion is important for visual navigation, predator avoidance and prey capture, and thus essential for the survival of all animals that have eyes. However, the direction of motion is not explicitly represented at the level of the photoreceptors: it rather needs to be computed by subsequent neural circuits, involving a comparison of the signals from neighboring photoreceptors over time. The exact nature of this process represents a classic example of neural computation and has been a longstanding question in the field. Much progress has been made in recent years in the fruit fly Drosophila melanogaster by genetically targeting individual neuron types to block, activate or record from them. Our results obtained this way demonstrate that the local direction of motion is computed in two parallel ON and OFF pathways. Within each pathway, a retinotopic array of four direction-selective T4 (ON) and T5 (OFF) cells represents the four Cartesian components of local motion vectors (leftward, rightward, upward, downward). Since none of the presynaptic neurons is directionally selective, direction selectivity first emerges within T4 and T5 cells. Our present research focuses on the cellular and biophysical mechanisms by which the direction of image motion is computed in these neurons.

How Intermittent Bioenergetic Challenges Enhance Brain and Body Health

Humans and other animals evolved in habitats fraught with a range of environmental challenges to their bodies and brains. Accordingly, cells and organ systems possess adaptive stress-responsive signaling pathways that enable them to not only withstand environmental challenges, but also to prepare for future challenges and function more efficiently. These phylogenetically conserved processes are the foundation of the hormesis principle in which repeated exposures to low to moderate amounts of an environmental challenge improve cellular and organismal fitness. Here I describe cellular and molecular mechanisms by which cells in the brain and body respond to intermittent fasting and exercise in ways that enhance performance and counteract aging and disease processes. Switching back and forth between adaptive stress response (during fasting and exercise) and growth and plasticity (eating, resting, sleeping) modes enhances the performance and resilience of various organ systems. While pharmacological interventions that engage a particular hormetic mechanism are being developed, it seems unlikely that any will prove superior to fasting and exercise.

In vivo direct imaging of neuronal activity at high temporospatial resolution

Advanced noninvasive neuroimaging methods provide valuable information on the brain function, but they have obvious pros and cons in terms of temporal and spatial resolution. Functional magnetic resonance imaging (fMRI) using blood-oxygenation-level-dependent (BOLD) effect provides good spatial resolution in the order of millimeters, but has a poor temporal resolution in the order of seconds due to slow hemodynamic responses to neuronal activation, providing indirect information on neuronal activity. In contrast, electroencephalography (EEG) and magnetoencephalography (MEG) provide excellent temporal resolution in the millisecond range, but spatial information is limited to centimeter scales. Therefore, there has been a longstanding demand for noninvasive brain imaging methods capable of detecting neuronal activity at both high temporal and spatial resolution. In this talk, I will introduce a novel approach that enables Direct Imaging of Neuronal Activity (DIANA) using MRI that can dynamically image neuronal spiking activity in milliseconds precision, achieved by data acquisition scheme of rapid 2D line scan synchronized with periodically applied functional stimuli. DIANA was demonstrated through in vivo mouse brain imaging on a 9.4T animal scanner during electrical whisker-pad stimulation. DIANA with milliseconds temporal resolution had high correlations with neuronal spike activities, which could also be applied in capturing the sequential propagation of neuronal activity along the thalamocortical pathway of brain networks. In terms of the contrast mechanism, DIANA was almost unaffected by hemodynamic responses, but was subject to changes in membrane potential-associated tissue relaxation times such as T2 relaxation time. DIANA is expected to break new ground in brain science by providing an in-depth understanding of the hierarchical functional organization of the brain, including the spatiotemporal dynamics of neural networks.

NOTE: DUE TO A CYBER ATTACK OUR UNIVERSITY WEB SYSTEM IS SHUT DOWN - TALK WILL BE RESCHEDULED

The size and structure of the dendritic arbor play important roles in determining how synaptic inputs of neurons are converted to action potential output and how neurons are integrated in the surrounding neuronal network. Accordingly, neurons with aberrant morphology have been associated with neurological disorders. Dysmorphic, enlarged neurons are, for example, a hallmark of focal epileptogenic lesions like focal cortical dysplasia (FCDIIb) and gangliogliomas (GG). However, the regulatory mechanisms governing the development of dendrites are insufficiently understood. The evolutionary conserved Ste20/Hippo kinase pathway has been proposed to play an important role in regulating the formation and maintenance of dendritic architecture. A key element of this pathway, Ste20-like kinase (SLK), regulates cytoskeletal dynamics in non-neuronal cells and is strongly expressed throughout neuronal development. Nevertheless, its function in neurons is unknown. We found that during development of mouse cortical neurons, SLK has a surprisingly specific role for proper elaboration of higher, ≥ 3rd, order dendrites both in cultured neurons and living mice. Moreover, SLK is required to maintain excitation-inhibition balance. Specifically, SLK knockdown causes a selective loss of inhibitory synapses and functional inhibition after postnatal day 15, while excitatory neurotransmission is unaffected. This mechanism may be relevant for human disease, as dysmorphic neurons within human cortical malformations exhibit significant loss of SLK expression. To uncover the signaling cascades underlying the action of SLK, we combined phosphoproteomics, protein interaction screens and single cell RNA seq. Overall, our data identifies SLK as a key regulator of both dendritic complexity during development and of inhibitory synapse maintenance.

Microbial modulation of zebrafish behavior and brain development

There is growing recognition that host-associated microbiotas modulate intrinsic neurodevelopmental programs including those underlying human social behavior. Despite this awareness, the fundamental processes are generally not understood. We discovered that the zebrafish microbiota is necessary for normal social behavior. By examining neuronal correlates of behavior, we found that the microbiota restrains neurite complexity and targeting of key forebrain neurons within the social behavior circuitry. The microbiota is also necessary for both localization and molecular functions of forebrain microglia, brain-resident phagocytes that remodel neuronal arbors. In particular, the microbiota promotes expression of complement signaling pathway components important for synapse remodeling. Our work provides evidence that the microbiota modulates zebrafish social behavior by stimulating microglial remodeling of forebrain circuits during early neurodevelopment and suggests molecular pathways for therapeutic interventions during atypical neurodevelopment.

Euclidean coordinates are the wrong prior for primate vision

The mapping from the visual field to V1 can be approximated by a log-polar transform. In this domain, scale is a left-right shift, and rotation is an up-down shift. When fed into a standard shift-invariant convolutional network, this provides scale and rotation invariance. However, translation invariance is lost. In our model, this is compensated for by multiple fixations on an object. Due to the high concentration of cones in the fovea with the dropoff of resolution in the periphery, fully 10 degrees of visual angle take up about half of V1, with the remaining 170 degrees (or so) taking up the other half. This layout provides the basis for the central and peripheral pathways. Simulations with this model closely match human performance in scene classification, and competition between the pathways leads to the peripheral pathway being used for this task. Remarkably, in spite of the property of rotation invariance, this model can explain the inverted face effect. We suggest that the standard method of using image coordinates is the wrong prior for models of primate vision.

Assigning credit through the "other” connectome

Learning in neural networks requires assigning the right values to thousands to trillions or more of individual connections, so that the network as a whole produces the desired behavior. Neuroscientists have gained insights into this “credit assignment” problem through decades of experimental, modeling, and theoretical studies. This has suggested key roles for synaptic eligibility traces and top-down feedback signals, among other factors. Here we study the potential contribution of another type of signaling that is being revealed in greater and greater fidelity by ongoing molecular and genomics studies. This is the set of modulatory pathways local to a given circuit, which form an intriguing second type of connectome overlayed on top of synaptic connectivity. We will share ongoing modeling and theoretical work that explores the possible roles of this local modulatory connectome in network learning.

The Neural Race Reduction: Dynamics of nonlinear representation learning in deep architectures

What is the relationship between task, network architecture, and population activity in nonlinear deep networks? I will describe the Gated Deep Linear Network framework, which schematizes how pathways of information flow impact learning dynamics within an architecture. Because of the gating, these networks can compute nonlinear functions of their input. We derive an exact reduction and, for certain cases, exact solutions to the dynamics of learning. The reduction takes the form of a neural race with an implicit bias towards shared representations, which then govern the model’s ability to systematically generalize, multi-task, and transfer. We show how appropriate network architectures can help factorize and abstract knowledge. Together, these results begin to shed light on the links between architecture, learning dynamics and network performance.

More than a beast growing in a passive brain: excitation and inhibition drive epilepsy and glioma progression

Gliomas are brain tumors formed by networks of connected tumor cells, nested in and interacting with neuronal networks. Neuronal activities interfere with tumor growth and occurrence of seizures affects glioma prognosis, while the developing tumor triggers seizures in the infiltrated cortex. Oncometabolites produced by tumor cells and neurotransmitters affect both the generation of epileptic activities by neurons and the growth of glioma cells through synaptic-related mechanisms, involving both GABAergic / Chloride pathways and glutamatergic signaling. From a clinical sight, epilepsy occurrence is beneficial to glioma prognosis but growing tumors are epileptogenic, which constitutes a paradox. This lecture will review how inhibitory and excitatory signaling drives glioma growth and how epileptic and oncological processes are interfering, with a special focus on the human brain.

The smart image compression algorithm in the retina: a theoretical study of recoding inputs in neural circuits

Computation in neural circuits relies on a common set of motifs, including divergence of common inputs to parallel pathways, convergence of multiple inputs to a single neuron, and nonlinearities that select some signals over others. Convergence and circuit nonlinearities, considered individually, can lead to a loss of information about the inputs. Past work has detailed how to optimize nonlinearities and circuit weights to maximize information, but we show that selective nonlinearities, acting together with divergent and convergent circuit structure, can improve information transmission over a purely linear circuit despite the suboptimality of these components individually. These nonlinearities recode the inputs in a manner that preserves the variance among converged inputs. Our results suggest that neural circuits may be doing better than expected without finely tuned weights.

Self-perception: mechanosensation and beyond

Brain-organ communications play a crucial role in maintaining the body's physiological and psychological homeostasis, and are controlled by complex neural and hormonal systems, including the internal mechanosensory organs. However, the progress has been slow due to technical hurdles: the sensory neurons are deeply buried inside the body and are not readily accessible for direct observation, the projection patterns from different organs or body parts are complex rather than converging into dedicate brain regions, the coding principle cannot be directly adapted from that learned from conventional sensory pathways. Our lab apply the pipeline of "biophysics of receptors-cell biology of neurons-functionality of neural circuits-animal behaviors" to explore the molecular and neural mechanisms of self-perception. In the lab, we mainly focus on the following three questions: 1, The molecular and cellular basis for proprioception and interoception. 2, The circuit mechanisms of sensory coding and integration of internal and external information. 3, The function of interoception in regulating behavior homeostasis.

Uncovering the molecular effectors of diet and exercise

Despite the profound effects of nutrition and physical activity on human health, our understanding of the molecules mediating the salutary effects of specific foods or activities remains remarkably limited. Here, we share our ongoing studies that use unbiased and high-resolution metabolomics technologies to uncover the molecules and molecular effectors of diet and exercise. We describe how exercise stimulates the production of Lac-Phe, a blood-borne signaling metabolite that suppresses feeding and obesity. Ablation of Lac-Phe biosynthesis in mice increases food intake and obesity after exercise. We also describe the discovery of an orphan metabolite, BHB-Phe. Ketosis-inducible BHB-Phe is a congener of exercise-inducible Lac-Phe, produced in CNDP2+ cells when levels of BHB are high, and functions to lower body weight and adiposity in ketosis. Our data uncover an unexpected and underappreciated signaling role for metabolic fuel derivatives in mediating the cardiometabolic benefits of diet and exercise. These data also suggest that diet and exercise may mediate their physiologic effects on energy balance via a common family of molecules and overlapping signaling pathways.

Integration of 3D human stem cell models derived from post-mortem tissue and statistical genomics to guide schizophrenia therapeutic development

Schizophrenia is a neuropsychiatric disorder characterized by positive symptoms (such as hallucinations and delusions), negative symptoms (such as avolition and withdrawal) and cognitive dysfunction1. Schizophrenia is highly heritable, and genetic studies are playing a pivotal role in identifying potential biomarkers and causal disease mechanisms with the hope of informing new treatments. Genome-wide association studies (GWAS) identified nearly 270 loci with a high statistical association with schizophrenia risk; however each locus confers only a small increase in risk therefore it is difficult to translate these findings into understanding disease biology that can lead to treatments. Induced pluripotent stem cell (iPSC) models are a tractable system to translate genetic findings and interrogate mechanisms of pathogenesis. Mounting research with patient-derived iPSCs has proposed several neurodevelopmental pathways altered in SCZ, such as neural progenitor cell (NPC) proliferation, imbalanced differentiation of excitatory and inhibitory cortical neurons. However, it is unclear what exactly these iPS models recapitulate, how potential perturbations of early brain development translates into illness in adults and how iPS models that represent fetal stages can be utilized to further drug development efforts to treat adult illness. I will present the largest transcriptome analysis of post-mortem caudate nucleus in schizophrenia where we discovered that decreased presynaptic DRD2 autoregulation is the causal dopamine risk factor for schizophrenia (Benjamin et al, Nature Neuroscience 2022 https://doi.org/10.1038/s41593-022-01182-7). We developed stem cell models from a subset of the postmortem cohort to better understand the molecular underpinnings of human psychiatric disorders (Sawada et al, Stem Cell Research 2020). We established a method for the differentiation of iPS cells into ventral forebrain organoids and performed single cell RNAseq and cellular phenotyping. To our knowledge, this is the first study to evaluate iPSC models of SZ from the same individuals with postmortem tissue. Our study establishes that striatal neurons in the patients with SCZ carry abnormalities that originated during early brain development. Differentiation of inhibitory neurons is accelerated whereas excitatory neuronal development is delayed, implicating an excitation and inhibition (E-I) imbalance during early brain development in SCZ. We found a significant overlap of genes upregulated in the inhibitory neurons in SCZ organoids with upregulated genes in postmortem caudate tissues from patients with SCZ compared with control individuals, including the donors of our iPS cell cohort. Altogether, we demonstrate that ventral forebrain organoids derived from postmortem tissue of individuals with schizophrenia recapitulate perturbed striatal gene expression dynamics of the donors’ brains (Sawada et al, biorxiv 2022 https://doi.org/10.1101/2022.05.26.493589).

Neuron-glial interactions in health and disease: from cognition to cancer

In the central nervous system, neuronal activity is a critical regulator of development and plasticity. Activity-dependent proliferation of healthy glial progenitors, oligodendrocyte precursor cells (OPCs), and the consequent generation of new oligodendrocytes contributes to adaptive myelination. This plasticity of myelin tunes neural circuit function and contributes to healthy cognition. The robust mitogenic effect of neuronal activity on normal oligodendroglial precursor cells, a putative cellular origin for many forms of glioma, suggests that dysregulated or “hijacked” mechanisms of myelin plasticity might similarly promote malignant cell proliferation in this devastating group of brain cancers. Indeed, neuronal activity promotes progression of both high-grade and low-grade glioma subtypes in preclinical models. Crucial mechanisms mediating activity-regulated glioma growth include paracrine secretion of BDNF and the synaptic protein neuroligin-3 (NLGN3). NLGN3 induces multiple oncogenic signaling pathways in the cancer cell, and also promotes glutamatergic synapse formation between neurons and glioma cells. Glioma cells integrate into neural circuits synaptically through neuron-to-glioma synapses, and electrically through potassium-evoked currents that are amplified through gap-junctional coupling between tumor cells This synaptic and electrical integration of glioma into neural circuits is central to tumor progression in preclinical models. Thus, neuron-glial interactions not only modulate neural circuit structure and function in the healthy brain, but paracrine and synaptic neuron-glioma interactions also play important roles in the pathogenesis of glial cancers. The mechanistic parallels between normal and malignant neuron-glial interactions underscores the extent to which mechanisms of neurodevelopment and plasticity are subverted by malignant gliomas, and the importance of understanding the neuroscience of cancer.

Impaired social reward valuation by chemogenetic inhibition of the primate prefronto-hypothalamic pathway

Altered dynamic information flow through the cortico-basal ganglia pathways is responsible for Parkinson’s disease symptoms

A microbiome-dependent gut-brain pathway regulates motivation for exercise

Myelin Formation and Oligodendrocyte Biology in Epilepsy

Epilepsy is one of the most common neurological diseases according to the World Health Organization (WHO) affecting around 70 million people worldwide [WHO]. Patients who suffer from epilepsy also suffer from a variety of neuro-psychiatric co-morbidities, which they can experience as crippling as the seizure condition itself. Adequate organization of cerebral white matter is utterly important for cognitive development. The failure of integration of neurologic function with cognition is reflected in neuro-psychiatric disease, such as autism spectrum disorder (ASD). However, in epilepsy we know little about the importance of white matter abnormalities in epilepsy-associated co-morbidities. Epilepsy surgery is an important therapy strategy in patients where conventional anti-epileptic drug treatment fails . On histology of the resected brain samples, malformations of cortical development (MCD) are common among the epilepsy surgery population, especially focal cortical dysplasia (FCD) and tuberous sclerosis complex (TSC). Both pathologies are associated with constitutive activation of the mTOR pathway. Interestingly, some type of FCD is morphological similar to TSC cortical tubers including the abnormalities of the white matter. Hypomyelination with lack of myelin-producing cells, the oligodendrocytes, within the lesional area is a striking phenomenon. Impairment of the complex myelination process can have a major impact on brain function. In the worst case leading to distorted or interrupted neurotransmissions. It is still unclear whether the observed myelin pathology in epilepsy surgical specimens is primarily related to the underlying malformation process or is just a secondary phenomenon of recurrent epileptic seizures creating a toxic micro-environment which hampers myelin formation. Interestingly, mTORC1 has been implicated as key signal for myelination, thus, promoting the maturation of oligodendrocytes . These results, however, remain controversial. Regardless of the underlying pathophysiologic mechanism, alterations of myelin dynamics, depending on their severity, are known to be linked to various kinds of developmental disorders or neuropsychiatric manifestations.

Brain mosaicism in epileptogenic cortical malformations

Focal Cortical Dysplasia (FCD) is the most common focal cortical malformation leading to intractable childhood focal epilepsy. In recent years, we and others have shown that FCD type II is caused by mosaic mutations in genes within the PI3K-AKT-mTOR-signaling pathway. Hyperactivation of the mTOR pathway accounts for neuropathological abnormalities and seizure occurrence in FCD. We further showed from human surgical FCDII tissue that epileptiform activity correlates with the density of mutated dysmorphic neurons, supporting their pro-epileptogenic role. The level of mosaicism, as defined by variant allele frequency (VAF) is thought to correlate with the size and regional brain distribution of the lesion such that when a somatic mutation occurs early during the cortical development, the dysplastic area is smaller than if it occurs later. Novel approaches based on the detection of cell-free DNA from the CSF and from trace tissue adherent to SEEG electrodes promise future opportunities for genetic testing during the presurgical evaluation of refractory epilepsy patients or in those that are not eligible for surgery. In utero-based electroporation mouse models allow to express somatic mutation during neurodevelopment and recapitulate most neuropathological and clinical features of FCDII, establishing relevant preclinical mouse models for developing precision medicine strategies.

Programmed axon death: from animal models into human disease

Programmed axon death is a widespread and completely preventable mechanism in injury and disease. Mouse and Drosophila studies define a molecular pathway involving activation of SARM1 NA Dase and its prevention by NAD synthesising enzyme NMNAT2 . Loss of axonal NMNAT2 causes its substrate, NMN , to accumulate and activate SARM1 , driving loss of NAD and changes in ATP , ROS and calcium. Animal models caused by genetic mutation, toxins, viruses or metabolic defects can be alleviated by blocking programmed axon death, for example models of CMT1B , chemotherapy-induced peripheral neuropathy (CIPN), rabies and diabetic peripheral neuropathy (DPN). The perinatal lethality of NMNAT2 null mice is completely rescued, restoring a normal, healthy lifespan. Animal models lack the genetic and environmental diversity present in human populations and this is problematic for modelling gene-environment combinations, for example in CIPN and DPN , and identifying rare, pathogenic mutations. Instead, by testing human gene variants in WGS datasets for loss- and gain-of-function, we identified enrichment of rare SARM1 gain-of-function variants in sporadic ALS , despite previous negative findings in SOD1 transgenic mice. We have shown in mice that heterozygous SARM1 loss-of-function is protective from a range of axonal stresses and that naturally-occurring SARM1 loss-of-function alleles are present in human populations. This enables new approaches to identify disorders where blocking SARM1 may be therapeutically useful, and the existence of two dominant negative human variants in healthy adults is some of the best evidence available that drugs blocking SARM1 are likely to be safe. Further loss- and gain-of-function variants in SARM1 and NMNAT2 are being identified and used to extend and strengthen the evidence of association with neurological disorders. We aim to identify diseases, and specific patients, in whom SARM1 -blocking drugs are most likely to be effective.

A possible role of the posterior alpha as a railroad switcher between dorsal and ventral pathways

Suppose you are on your favorite touchscreen device consciously and deliberately deciding emails to read or delete. In other words, you are consciously and intentionally looking, tapping, and swiping. Now suppose that you are doing this while neuroscientists are recording your brain activity. Eventually, the neuroscientists are familiar enough with your brain activity and behavior that they run an experiment with subliminal cues which reveals that your looking, tapping, and swiping seem to be determined by a random switch in your brain. You are not aware of it, or its impact on your decisions or movements. Would these predictions undermine your sense of free will? Some have argued that it should. Although this inference from unreflective and/or random intention mechanisms to free will skepticism, may seem intuitive at first, there are already objections to it. So, even if this thought experiment is plausible, it may not actually undermine our sense of free will.

Geometry of concept learning

Understanding Human ability to learn novel concepts from just a few sensory experiences is a fundamental problem in cognitive neuroscience. I will describe a recent work with Ben Sorcher and Surya Ganguli (PNAS, October 2022) in which we propose a simple, biologically plausible, and mathematically tractable neural mechanism for few-shot learning of naturalistic concepts. We posit that the concepts that can be learned from few examples are defined by tightly circumscribed manifolds in the neural firing-rate space of higher-order sensory areas. Discrimination between novel concepts is performed by downstream neurons implementing ‘prototype’ decision rule, in which a test example is classified according to the nearest prototype constructed from the few training examples. We show that prototype few-shot learning achieves high few-shot learning accuracy on natural visual concepts using both macaque inferotemporal cortex representations and deep neural network (DNN) models of these representations. We develop a mathematical theory that links few-shot learning to the geometric properties of the neural concept manifolds and demonstrate its agreement with our numerical simulations across different DNNs as well as different layers. Intriguingly, we observe striking mismatches between the geometry of manifolds in intermediate stages of the primate visual pathway and in trained DNNs. Finally, we show that linguistic descriptors of visual concepts can be used to discriminate images belonging to novel concepts, without any prior visual experience of these concepts (a task known as ‘zero-shot’ learning), indicated a remarkable alignment of manifold representations of concepts in visual and language modalities. I will discuss ongoing effort to extend this work to other high level cognitive tasks.

Social attention & emotion: invasive neurophysiology & white matter pathway studies

Microglial efferocytosis: Diving into the Alzheimer's Disease gene pool

Genome-wide association studies and functional genomics studies have linked specific cell types, genes, and pathways to Alzheimer’s disease (AD) risk. In particular, AD risk alleles primarily affect the abundance or structure, and thus the activity, of genes expressed in macrophages, strongly implicating microglia (the brain-resident macrophages) in the etiology of AD. These genes converge on pathways (endocytosis/phagocytosis, cholesterol metabolism, and immune response) with critical roles in core macrophage functions such as efferocytosis. Here, we review these pathways, highlighting relevant genes identified in the latest AD genetics and genomics studies, and describe how they may contribute to AD pathogenesis. Investigating the functional impact of AD-associated variants and genes in microglia is essential for elucidating disease risk mechanisms and developing effective therapeutic approaches." https://doi.org/10.1016/j.neuron.2022.10.015

Cholesterol and matrisome pathways dysregulated in Alzheimer’s disease brain astrocytes and microglia

The impact of apolipoprotein E ε4 (APOE4), the strongest genetic risk factor for Alzheimer’s disease (AD), on human brain cellular function remains unclear. Here, we investigated the effects of APOE4 on brain cell types derived from population and isogenic human induced pluripotent stem cells, post-mortem brain, and APOE targeted replacement mice. Population and isogenic models demonstrate that APOE4 local haplotype, rather than a single risk allele, contributes to risk. Global transcriptomic analyses reveal human-specific, APOE4-driven lipid metabolic dysregulation in astrocytes and microglia. APOE4 enhances de novo cholesterol synthesis despite elevated intracellular cholesterol due to lysosomal cholesterol sequestration in astrocytes. Further, matrisome dysregulation is associated with upregulated chemotaxis, glial activation, and lipid biosynthesis in astrocytes co-cultured with neurons, which recapitulates altered astrocyte matrisome signaling in human brain. Thus, APOE4 initiates glia-specific cell and non-cell autonomous dysregulation that may contribute to increased AD risk." https://doi.org/10.1016/j.cell.2022.05.017

Prefrontal top-down projections control context-dependent strategy selection

The rules governing behavior often vary with behavioral contexts. As a result, an action rewarded in one context may be discouraged in another. Animals and humans are capable of switching between behavioral strategies under different contexts and acting adaptively according to the variable rules, a flexibility that is thought to be mediated by the prefrontal cortex (PFC). However, how the PFC orchestrates the context-dependent switch of strategies remains unclear. Here we show that pathway-specific projection neurons in the medial PFC (mPFC) differentially contribute to context-instructed strategy selection. In mice trained in a decision-making task in which a previously established rule and a newly learned rule are associated with distinct contexts, the activity of mPFC neurons projecting to the dorsomedial striatum (mPFC-DMS) encodes the contexts and further represents decision strategies conforming to the old and new rules. Moreover, mPFC-DMS neuron activity is required for the context-instructed strategy selection. In contrast, the activity of mPFC neurons projecting to the ventral midline thalamus (mPFC-VMT) does not discriminate between the contexts, and represents the old rule even if mice have adopted the new one. Furthermore, these neurons act to prevent the strategy switch under the new rule. Our results suggest that mPFC-DMS neurons promote flexible strategy selection guided by contexts, whereas mPFC-VMT neurons favor fixed strategy selection by preserving old rules.

Shallow networks run deep: How peripheral preprocessing facilitates odor classification

Drosophila olfactory sensory hairs ("sensilla") typically house two olfactory receptor neurons (ORNs) which can laterally inhibit each other via electrical ("ephaptic") coupling. ORN pairing is highly stereotyped and genetically determined. Thus, olfactory signals arriving in the Antennal Lobe (AL) have been pre-processed by a fixed and shallow network at the periphery. To uncover the functional significance of this organization, we developed a nonlinear phenomenological model of asymmetrically coupled ORNs responding to odor mixture stimuli. We derived an analytical solution to the ORNs’ dynamics, which shows that the peripheral network can extract the valence of specific odor mixtures via transient amplification. Our model predicts that for efficient read-out of the amplified valence signal there must exist specific patterns of downstream connectivity that reflect the organization at the periphery. Analysis of AL→Lateral Horn (LH) fly connectomic data reveals evidence directly supporting this prediction. We further studied the effect of ephaptic coupling on olfactory processing in the AL→Mushroom Body (MB) pathway. We show that stereotyped ephaptic interactions between ORNs lead to a clustered odor representation of glomerular responses. Such clustering in the AL is an essential assumption of theoretical studies on odor recognition in the MB. Together our work shows that preprocessing of olfactory stimuli by a fixed and shallow network increases sensitivity to specific odor mixtures, and aids in the learning of novel olfactory stimuli. Work led by Palka Puri, in collaboration with Chih-Ying Su and Shiuan-Tze Wu.

How fly neurons compute the direction of visual motion

Detecting the direction of image motion is important for visual navigation, predator avoidance and prey capture, and thus essential for the survival of all animals that have eyes. However, the direction of motion is not explicitly represented at the level of the photoreceptors: it rather needs to be computed by subsequent neural circuits. The exact nature of this process represents a classic example of neural computation and has been a longstanding question in the field. Our results obtained in the fruit fly Drosophila demonstrate that the local direction of motion is computed in two parallel ON and OFF pathways. Within each pathway, a retinotopic array of four direction-selective T4 (ON) and T5 (OFF) cells represents the four Cartesian components of local motion vectors (leftward, rightward, upward, downward). Since none of the presynaptic neurons is directionally selective, direction selectivity first emerges within T4 and T5 cells. Our present research focuses on the cellular and biophysical mechanisms by which the direction of image motion is computed in these neurons.

Restructuring cortical feedback circuits

We hardly notice when there is a speck on our glasses, the obstructed visual information seems to be magically filled in. The mechanistic basis for this fundamental perceptual phenomenon has, however, remained obscure. What enables neurons in the visual system to respond to context when the stimulus is not available? While feedforward information drives the activity in cortex, feedback information is thought to provide contextual signals that are merely modulatory. We have made the discovery that mouse primary visual cortical neurons are strongly driven by feedback projections from higher visual areas when their feedforward sensory input from the retina is missing. This drive is so strong that it makes visual cortical neurons fire as much as if they were receiving a direct sensory input. These signals are likely used to predict input from the feedforward pathway. Preliminary results show that these feedback projections are strongly influenced by experience and learning.

Myelin Formation and Oligodendrocyte Biology in Epilepsy

Epilepsy is one of the most common neurological diseases according to the World Health Organization (WHO) affecting around 70 million people worldwide [WHO]. Patients who suffer from epilepsy also suffer from a variety of neuro-psychiatric co-morbidities, which they can experience as crippling as the seizure condition itself. Adequate organization of cerebral white matter is utterly important for cognitive development. The failure of integration of neurologic function with cognition is reflected in neuro-psychiatric disease, such as autism spectrum disorder (ASD). However, in epilepsy we know little about the importance of white matter abnormalities in epilepsy-associated co-morbidities. Epilepsy surgery is an important therapy strategy in patients where conventional anti-epileptic drug treatment fails . On histology of the resected brain samples, malformations of cortical development (MCD) are common among the epilepsy surgery population, especially focal cortical dysplasia (FCD) and tuberous sclerosis complex (TSC). Both pathologies are associated with constitutive activation of the mTOR pathway. Interestingly, some type of FCD is morphological similar to TSC cortical tubers including the abnormalities of the white matter. Hypomyelination with lack of myelin-producing cells, the oligodendrocytes, within the lesional area is a striking phenomenon. Impairment of the complex myelination process can have a major impact on brain function. In the worst case leading to distorted or interrupted neurotransmissions. It is still unclear whether the observed myelin pathology in epilepsy surgical specimens is primarily related to the underlying malformation process or is just a secondary phenomenon of recurrent epileptic seizures creating a toxic micro-environment which hampers myelin formation. Interestingly, mTORC1 has been implicated as key signal for myelination, thus, promoting the maturation of oligodendrocytes . These results, however, remain controversial. Regardless of the underlying pathophysiologic mechanism, alterations of myelin dynamics, depending on their severity, are known to be linked to various kinds of developmental disorders or neuropsychiatric manifestations.

Lateral entorhinal cortex directly influences medial entorhinal cortex through synaptic connections in layer 1

Standard models of episodic memory suggest that lateral (LEC) and medial entorhinal cortex (MEC) send independent inputs to the hippocampus, each carrying different types of information. Here, we describe a pathway by which information is integrated between LEC and MEC prior to reaching hippocampus. We demonstrate that LEC sends strong projections to MEC arising from neurons that receive neocortical inputs. Activation of LEC inputs drives excitation of hippocampal-projecting neurons in MEC layer 2, typically followed by inhibition that is accounted for by parallel activation of local inhibitory neurons. We therefore propose that local circuits in MEC may support integration of ‘what’ and ‘where’ information.

Inter-tissue signals modify food-seeking behavior in C. elegans

Animals modify their behavioral outputs in response to changes in external and internal environments. We use the nematode, C. elegans to probe the pathways linking changes in internal states like hunger with behavior. We find that acute food deprivation alters the localization of two transcription factors, likely releasing an insulin-like peptide from the intestine, which in turn modifies chemosensory neurons and alters behavior. These results present a model for how inter-tissue signals to generate flexible behaviors via gut-brain signaling.

Redox and mitochondrial dysregulation in epilepsy

Epileptic seizures render the brain uniquely dependent on energy producing pathways. Studies in our laboratory have been focused on the role of redox processes and mitochondria in the context of abnormal neuronal excitability associated with epilepsy. We have shown that that status epilepticus (SE) alters mitochondrial and cellular redox status, energetics and function and conversely, that reactive oxygen species and resultant dysfunction can lead to chronic epilepsy. Oxidative stress and neuroinflammatory pathways have considerable crosstalk and targeting redox processes has recently been shown to control neuroinflammation and excitability. Understanding the role of metabolic and redox processes can enable the development of novel therapeutics to control epilepsy and/or its comorbidities.

IMMUNOHISTOCHEMICAL CHARACTERIZATION OF THE RETINA–OPTIC NERVE–BRAIN VISUAL PATHWAY IN THE AFRICAN TREE SQUIRREL

FENS Forum 2026

Investigating the role of recurrent connectivity in connectome-constrained and task-optimized models of the fruit fly’s motion pathway

Bernstein Conference 2024

Cell-type specific electrophysiological and behavioural alterations in the globus pallidus of mice following striatopallidal pathway ablation

A visuomotor pathway underlies small object avoidance in flying Drosophila

COSYNE 2022

A visuomotor pathway underlies small object avoidance in flying Drosophila

COSYNE 2022

Efficient connectome analyses for identifying bottleneck neurons in behaviorally-relevant pathways

COSYNE 2023

Activity-driven deep models for learning sound transformations across the auditory pathway

Changes in tuning curves, not neural population covariance, improve category separability in the primate ventral visual pathway

COSYNE 2025

An Efficient Multilayer Spiking Network as a Model of Ascending Pathways

COSYNE 2025

A prefrontal to amygdala pathway for adaptive sleep interruption

COSYNE 2025

Stimulus-specific contributions of cortical and collicular pathways to visual feature detection

COSYNE 2025

Striatal pathways oppositely shift cortical activity along the decision axis

COSYNE 2025

Activation of the hepcidin-ferroportin1 pathway in the brain and astrocytic-neuronal crosstalk to counteract iron dyshomeostasis during aging

Activation of regeneration-associated pathways in neurons following photocapacitive stimulation

Mechanisms of contextual fear memory suppression and extinction by the Nucleus Reuniens-CA1 pathway

COSYNE 2023

Ageing-associated alterations in BDNF/TrkB pathway involving PKC and synaptic targets of the Neuromuscular Junction

Alogliptin Attenuates Lipopolysaccharide-Induced Neuroinflammation in Mice Through Modulation of TLR4/MYD88/NF-κB and miRNA-155/SOCS-1 Signaling Pathways

Alterations of kynurenine pathway in peripheral and central nervous system under hypobaric hypoxic stress

Altered expression of peripheral microRNAs and pathway signaling after mild traumatic brain injury

Antidepressant actions of ketamine engage cellular mechanisms of endoplasmic reticulum stress by the eIF2α pathway

The ApoE ε4 genetic polymorphism alters cholesterol metabolism and cholinergic signalling pathway promoting neurotoxic effects

Association of canonical NF-κB signaling pathway with apoptotic cell death and cell proliferation in glucocorticoid-induced neurotoxicity and after vitamin D3 supplementation

Asymmetric encoding dynamics in the striatal direct and indirect pathways during spontaneous behaviour

Axonal vesicles are pentose phosphate pathway mobile platforms crucial for ROS detoxification and neuronal survival

Basal Ganglia-Spinal Cord Pathway that Commands Locomotor Asymmetries

The basolateral amygdala to nucleus accumbens shell pathway encodes, but doesn’t retrieve, outcome-specific predictions to guide choice between actions

BDNF/TrkB pathway activation in D1 receptor-expressing striatal projection neurons plays a protective role against L-DOPA-induced dyskinesia

Blocking the P2X7-NLRP3-IL-1β pathway in the maternal immune activation model prevents autism-like phenotype in male mouse offspring

Bridging collaterals act in concert with the canonical basal ganglia direct pathway to support motor control

A Cav1.4 L-type calcium channel truncation mutation affects the retinal rod pathway

Dual pathway architecture in songbirds boosts sensorimotor learning

COSYNE 2022

Characterising ‘the munchies’; effects of tetrahydrocannabinol (THC) vapour inhalation on rat feeding behaviours and homeostatic appetite-regulating pathways

Characterization of anatomical and functional vestibulospinal pathways in larval Xenopus laevis

Characterizing the utility of novel channelrhodopsin mutants for activation of the auditory pathway

The connection between primary cilia and the hypoxia-inducible factor-2alpha promotes the MEK/ERK signaling pathway

Constitutive 5-HT2C receptor knock-out facilitates fear extinction through altered activity of a dorsal raphe–bed nucleus of the stria terminalis pathway

Contribution of GBA1 mutations to autophagy-lysosomal pathway in Parkinson's disease

Cortico-hippocampal bilateral coherence is variable and pathway-specific: a study with field potential generators

CRMP4-mediated fornix development involves semaphorin-3E signaling pathway

Dual role, single pathway: A pyramidal cell model of feedback integration in function and learning

Bernstein Conference 2024